HUMANIZED AND AFFINITY MATURED ANTIBODIES TO FcRH5 AND METHODS OF USE

ABSTRACT

The present invention relates to anti-FcRH5 antibodies, including anti-FcRH5 antibodies comprising an FcRH5 binding domain and a CD3 binding domain (e.g., FcRH5 T cell-dependent bispecific (TDB) antibodies), and methods of using the same.

FIELD OF THE INVENTION

The present invention relates to anti-FcRH5 antibodies and methods ofusing the same.

BACKGROUND OF THE INVENTION

Cell proliferative disorders, such as cancer, are characterized by theuncontrolled growth of cell subpopulations. They are the leading causeof death in the developed world and the second leading cause of death indeveloping countries, with over 14 million new cancer cases diagnosedand over eight million cancer deaths occurring each year. The NationalCancer Institute has estimated that greater than half a millionAmericans will die of cancer in 2016, accounting for nearly one out ofevery four deaths in the country. As the elderly population has grown,the incidence of cancer has concurrently risen, as the probability ofdeveloping cancer is more than two-fold higher after the age of seventy.Cancer care thus represents a significant and ever-increasing societalburden.

The Fc receptor-like 5 (FcRH5, also known as FcRL5 or IRTA2) genebelongs to a family of six recently identified genes of theimmunoglobulin superfamily (IgSF). This family of genes is closelyrelated to the Fc receptors with the conserved genomic structure,extracellular Ig domain composition, and immunoreceptor tyrosine-basedinhibitory (ITIM) and immunoreceptor tyrosine-based activation (ITAM)like signaling motifs (Davis et al. Eur. J. Immunol. 35:674-80, 2005).Six members of the FcRH/IRTA receptor family have been described:FcRH1/IRTA5, FcRH2/IRTA4, FcRH3/IRTA3, FcRH4/IRTA1, FcRH5/IRTA2, andFcRH6 (Polson et al. Int. Immunol. 18(9):1363-1373, 2006. The FcRH cDNAsencode type I transmembrane glycoproteins with multiple Ig-likeextracellular domains and cytoplasmic domains containing consensusimmunoreceptor tyrosine-based activating and/or inhibitory signalingmotifs. The FcRH genes are structurally related, and their proteinproducts share 28-60% extracellular identity with each other. They alsoshare 15-31% identity with their closest FcR relatives. There is a highdegree of homology between the different FcRHs.

The ligand(s) for FcRH5 are unknown, but FcRH5 has been implicated inenhanced proliferation and downstream isotype expression during thedevelopment of antigen-primed B-cells (Dement-Brown et al. J. Leukoc.Biol. 91:59-67, 2012). The FcRH5 locus has three major mRNA isoforms(FcRH5a, FcRH5b, and FcRH5c). The major FcRH5 protein isoforms encodedby these transcripts share a common amino acid sequence until residue560, featuring a common signal peptide and six extracellular Ig-likedomains. FcRH5a represents a 759-amino acid secreted glycoprotein witheight Ig-like domains followed by 13 unique, predominantly polar aminoacids at its C-terminus. FcRH5b diverges from FcRH5a at amino acidresidue 560 and extends for a short stretch of 32 additional residues,whose hydrophobicity is compatible with its docking to the plasmamembrane via a GPI anchor. FcRH5c is the longest isoform whose sequencedeviates from FcRH5a at amino acid 746. FcRH5c encodes a 977-amino acidtype I transmembrane glycoprotein with nine extracellular Ig-typedomains harboring eight potential N-linked glycosylation sites, a23-amino acid transmembrane domain, and a 104-amino acid cytoplasmicdomain with three consensus SH2 binding motifs having an ITIM consensus.

The FcRH genes are clustered together in the midst of the classical FcRgenes (FcγRI, FcγRII, FcγRIII, and FcRI) in the 1q21-23 region ofchromosome 1. This region contains one of the most frequent secondarychromosomal abnormalities associated with malignant phenotype inhematopoietic tumors, especially in multiple myeloma (Hatzivassiliou etal. Immunity. 14:277-89, 2001). FcRH5 is expressed only in the B-celllineage, starting as early as pre-B-cells, but does not attain fullexpression until the mature B-cell stage. Unlike most known otherB-cell-specific surface proteins (e.g., CD20, CD19, and CD22), FcRH5continues to be expressed in plasma cells, whereas other B-cell-specificmarkers are downregulated (Polson et al. Int. Immunol. 18:1363-73,2006). In addition, FcRH5 mRNA is overexpressed in multiple myeloma celllines with 1q21 abnormalities as detected by oligonucleotide arrays(Inoue Am. J. Pathol. 165:71-81, 2004). The expression pattern indicatesthat FcRH5 could be a target for antibody-based therapies for thetreatment of multiple myeloma. Multiple myeloma is a malignancy ofplasma cells characterized by skeletal lesions, renal failure, anemia,and hypercalcemia, and it is essentially incurable by current therapies.Current drug treatments for multiple myeloma include combinations of theproteosome inhibitor bortezomib (VELCADE®), the immunomodulatorlenalidomide (REVLIMID®), and the steroid dexamethasone.

Monoclonal antibody (mAb)-based therapy has become an importanttreatment modality for cancer. FcRH5c-specific antibody-based therapiesand detection methods may be particularly efficacious as theyspecifically recognize target cell, membrane-associated FcRH5 ratherthan antibodies which recognize both soluble and membrane isoforms ofFcRH5. However, only the last Ig-like domain of FcRH5 (Ig-like domain 9)is a unique extracellular region that differentiates between the threemajor isoforms of FcRH5 (e.g., FcRH5a, FcRH5b, and FcRH5c), and there issignificant homology between the Ig-like domains within FcRH5. Further,the last Ig-like domain is highly conserved between FcRH1, FcRH2, FcRH3,and FcRH5. Any antibody-based therapy that specifically targeted FcRH5should have minimal cross-reactivity with other FcRHs to avoid adverseoff-target effects (e.g., FcRH3 is expressed on normal NK cells).

In view of the above, there is an unmet need in the field for safe andeffective agents for use in the treatment of cell proliferativedisorders (e.g., cancers, e.g., FcRH5-positive cancers, e.g., multiplemyeloma).

SUMMARY OF THE INVENTION

The present invention provides anti-FcRH5 antibodies (e.g., bispecificantibodies, e.g., FcRH5 T cell-dependant bispecific (TDB) antibodies),compositions, and methods of using the same for the treatment of cellproliferative disorders (e.g., cancers, e.g., FcRH5-positive cancers,e.g., multiple myeloma).

In a first aspect, the invention features an anti-Fc Receptor-like 5(FcRH5) antibody, including a binding domain comprising the followingsix hypervariable regions (HVRs): (a) an HVR-H1 comprising the aminoacid sequence of SEQ ID NO: 1, (b) an HVR-H2 comprising the amino acidsequence of SEQ ID NO: 2, (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 3, (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 4, (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 5, and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 6.

In some embodiments, the anti-FcRH5 antibody comprises a binding domaincomprising the following six HVRs: (a) an HVR-H1 comprising the aminoacid sequence of SEQ ID NO: 1, (b) an HVR-H2 comprising the amino acidsequence of SEQ ID NO: 8, (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 9, (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 12, (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 16, and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 23. In some embodiments, the binding domaincomprises (a) a heavy chain variable (VH) domain comprising an aminoacid sequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO: 104, (b) a light chain variable (VL) domaincomprising an amino acid sequence having at least 95% sequence identityto the amino acid sequence of SEQ ID NO: 105, or (c) a VH domain as in(a) and a VL domain as in (b). In some embodiments, the antibody furthercomprises the following heavy chain variable region framework regions(FRs): (a) an FR-H1 comprising the amino acid sequence of SEQ ID NO: 52,(b) an FR-H2 comprising the amino acid sequence of SEQ ID NO: 54, (c) anFR-H3 comprising the amino acid sequence of SEQ ID NO: 46, and (d) anFR-H4 comprising the amino acid sequence of SEQ ID NO: 47. In someembodiments, the VH domain comprises the amino acid sequence of SEQ IDNO: 104. In some embodiments, the antibody further comprises thefollowing light chain variable region FRs: (a) an FR-L1 comprising theamino acid sequence of SEQ ID NO: 48, (b) an FR-L2 comprising the aminoacid sequence of SEQ ID NO: 57, (c) an FR-L3 comprising the amino acidsequence of SEQ ID NO: 50, and (d) an FR-L4 comprising the amino acidsequence of SEQ ID NO: 51. In some embodiments, the VL domain comprisesthe amino acid sequence of SEQ ID NO: 105. In some embodiments, theanti-FcRH5 antibody comprises a binding domain comprising (a) a VHdomain comprising an amino acid sequence of SEQ ID NO: 104 and (b) a VLdomain comprising an amino acid sequence of SEQ ID NO: 105.

In other embodiments, the anti-FcRH5 antibody comprises a binding domaincomprising the following six HVRs: (a) an HVR-H1 comprising the aminoacid sequence of SEQ ID NO: 1, (b) an HVR-H2 comprising the amino acidsequence of SEQ ID NO: 8, (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 10, (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 14, (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 16, and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 23. In some embodiments, the binding domaincomprises (a) a VH domain comprising an amino acid sequence having atleast 95% sequence identity to the amino acid sequence of SEQ ID NO:106, (b) a VL domain comprising an amino acid sequence having at least95% sequence identity to the amino acid sequence of SEQ ID NO: 107, or(c) a VH domain as in (a) and a VL domain as in (b). In someembodiments, the antibody further comprises the following heavy chainvariable region FRs: (a) an FR-H1 comprising the amino acid sequence ofSEQ ID NO: 53, (b) an FR-H2 comprising the amino acid sequence of SEQ IDNO: 54, (c) an FR-H3 comprising the amino acid sequence of SEQ ID NO:46, and (d) an FR-H4 comprising the amino acid sequence of SEQ ID NO:47. In some embodiments, the VH domain comprises the amino acid sequenceof SEQ ID NO: 106. In some embodiments, the antibody further comprisesthe following light chain variable region FRs: (a) an FR-L1 comprisingthe amino acid sequence of SEQ ID NO: 48, (b) an FR-L2 comprising theamino acid sequence of SEQ ID NO: 57, (c) an FR-L3 comprising the aminoacid sequence of SEQ ID NO: 50, and (d) an FR-L4 comprising the aminoacid sequence of SEQ ID NO: 51. In some embodiments, the VL domaincomprises the amino acid sequence of SEQ ID NO: 107. In someembodiments, the anti-FcRH5 antibody comprises a binding domaincomprising (a) a VH domain comprising an amino acid sequence of SEQ IDNO: 106 and (b) a VL domain comprising an amino acid sequence of SEQ IDNO: 107.

In other embodiments, the anti-FcRH5 antibody comprises a binding domaincomprising the following six HVRs: (a) an HVR-H1 comprising the aminoacid sequence of SEQ ID NO: 1, (b) an HVR-H2 comprising the amino acidsequence of SEQ ID NO: 7, (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 9, (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 11, (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 15, and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 20. In some embodiments, the binding domaincomprises (a) a VH domain comprising an amino acid sequence having atleast 95% sequence identity to the amino acid sequence of SEQ ID NO: 82,(b) a VL domain comprising an amino acid sequence having at least 95%sequence identity to the amino acid sequence of SEQ ID NO: 83, or (c) aVH domain as in (a) and a VL domain as in (b). In some embodiments, theantibody further comprises the following heavy chain variable regionFRs: (a) an FR-H1 comprising the amino acid sequence of SEQ ID NO: 52,(b) an FR-H2 comprising the amino acid sequence of SEQ ID NO: 54, (c) anFR-H3 comprising the amino acid sequence of SEQ ID NO: 46, and (d) anFR-H4 comprising the amino acid sequence of SEQ ID NO: 47. In someembodiments, the VH domain comprises the amino acid sequence of SEQ IDNO: 82. In some embodiments, the antibody further comprises thefollowing light chain variable region FRs: (a) an FR-L1 comprising theamino acid sequence of SEQ ID NO: 48, (b) an FR-L2 comprising the aminoacid sequence of SEQ ID NO: 56, (c) an FR-L3 comprising the amino acidsequence of SEQ ID NO: 50, and (d) an FR-L4 comprising the amino acidsequence of SEQ ID NO: 51. In some embodiments, the VL domain comprisesthe amino acid sequence of SEQ ID NO: 83. In some embodiments, theanti-FcRH5 antibody comprises a binding domain comprising (a) a VHdomain comprising an amino acid sequence of SEQ ID NO: 82 and (b) a VLdomain comprising an amino acid sequence of SEQ ID NO: 83.

In other embodiments, the anti-FcRH5 antibody comprises a binding domaincomprising the following six HVRs: (a) an HVR-H1 comprising the aminoacid sequence of SEQ ID NO: 1, (b) an HVR-H2 comprising the amino acidsequence of SEQ ID NO: 7, (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 9, (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 12, (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 16, and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 21. In some embodiments, the binding domaincomprises (a) a VH domain comprising an amino acid sequence having atleast 95% sequence identity to the amino acid sequence of SEQ ID NO: 84,(b) a VL domain comprising an amino acid sequence having at least 95%sequence identity to the amino acid sequence of SEQ ID NO: 85, or (c) aVH domain as in (a) and a VL domain as in (b). In some embodiments, theantibody further comprises the following heavy chain variable regionFRs: (a) an FR-H1 comprising the amino acid sequence of SEQ ID NO: 52,(b) an FR-H2 comprising the amino acid sequence of SEQ ID NO: 54, (c) anFR-H3 comprising the amino acid sequence of SEQ ID NO: 46, and (d) anFR-H4 comprising the amino acid sequence of SEQ ID NO: 47. In someembodiments, the VH domain comprises the amino acid sequence of SEQ IDNO: 84. In some embodiments, the antibody further comprises thefollowing light chain variable region FRs: (a) an FR-L1 comprising theamino acid sequence of SEQ ID NO: 48, (b) an FR-L2 comprising the aminoacid sequence of SEQ ID NO: 57, (c) an FR-L3 comprising the amino acidsequence of SEQ ID NO: 50, and (d) an FR-L4 comprising the amino acidsequence of SEQ ID NO: 51. In some embodiments, the VL domain comprisesthe amino acid sequence of SEQ ID NO: 85. In some embodiments, theanti-FcRH5 antibody comprises a binding domain comprising (a) a VHdomain comprising an amino acid sequence of SEQ ID NO: 84 and (b) a VLdomain comprising an amino acid sequence of SEQ ID NO: 85.

In other embodiments, the anti-FcRH5 antibody comprises a binding domaincomprising the following six HVRs: (a) an HVR-H1 comprising the aminoacid sequence of SEQ ID NO: 1, (b) an HVR-H2 comprising the amino acidsequence of SEQ ID NO: 7, (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 9, (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 12, (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 17, and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 22. In some embodiments, the binding domaincomprises (a) a VH domain comprising an amino acid sequence having atleast 95% sequence identity to the amino acid sequence of SEQ ID NO: 86,(b) a VL domain comprising an amino acid sequence having at least 95%sequence identity to the amino acid sequence of SEQ ID NO: 87, or (c) aVH domain as in (a) and a VL domain as in (b). In some embodiments, theantibody further comprises the following heavy chain variable regionFRs: (a) an FR-H1 comprising the amino acid sequence of SEQ ID NO: 52,(b) an FR-H2 comprising the amino acid sequence of SEQ ID NO: 54, (c) anFR-H3 comprising the amino acid sequence of SEQ ID NO: 46, and (d) anFR-H4 comprising the amino acid sequence of SEQ ID NO: 47. In someembodiments, the VH domain comprises the amino acid sequence of SEQ IDNO: 86. In some embodiments, the antibody further comprises thefollowing light chain variable region FRs: (a) an FR-L1 comprising theamino acid sequence of SEQ ID NO: 48, (b) an FR-L2 comprising the aminoacid sequence of SEQ ID NO: 57, (c) an FR-L3 comprising the amino acidsequence of SEQ ID NO: 50, and (d) an FR-L4 comprising the amino acidsequence of SEQ ID NO: 51. In some embodiments, the VL domain comprisesthe amino acid sequence of SEQ ID NO: 87. In some embodiments, theanti-FcRH5 antibody comprises a binding domain comprising (a) a VHdomain comprising an amino acid sequence of SEQ ID NO: 86 and (b) a VLdomain comprising an amino acid sequence of SEQ ID NO: 87.

In other embodiments, the anti-FcRH5 antibody comprises a binding domaincomprising the following six HVRs: (a) an HVR-H1 comprising the aminoacid sequence of SEQ ID NO: 1, (b) an HVR-H2 comprising the amino acidsequence of SEQ ID NO: 7, (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 9, (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 13, (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 16, and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 21. In some embodiments, the binding domaincomprises (a) a VH domain comprising an amino acid sequence having atleast 95% sequence identity to the amino acid sequence of SEQ ID NO: 88,(b) a VL domain comprising an amino acid sequence having at least 95%sequence identity to the amino acid sequence of SEQ ID NO: 89, or (c) aVH domain as in (a) and a VL domain as in (b). In some embodiments, theantibody further comprises the following heavy chain variable regionFRs: (a) an FR-H1 comprising the amino acid sequence of SEQ ID NO: 52,(b) an FR-H2 comprising the amino acid sequence of SEQ ID NO: 54, (c) anFR-H3 comprising the amino acid sequence of SEQ ID NO: 46, and (d) anFR-H4 comprising the amino acid sequence of SEQ ID NO: 47. In someembodiments, the VH domain comprises the amino acid sequence of SEQ IDNO: 88. In some embodiments, the antibody further comprises thefollowing light chain variable region FRs: (a) an FR-L1 comprising theamino acid sequence of SEQ ID NO: 48, (b) an FR-L2 comprising the aminoacid sequence of SEQ ID NO: 57, (c) an FR-L3 comprising the amino acidsequence of SEQ ID NO: 50, and (d) an FR-L4 comprising the amino acidsequence of SEQ ID NO: 51. In some embodiments, the VL domain comprisesthe amino acid sequence of SEQ ID NO: 89. In some embodiments, theanti-FcRH5 antibody comprises a binding domain comprising (a) a VHdomain comprising an amino acid sequence of SEQ ID NO: 88 and (b) a VLdomain comprising an amino acid sequence of SEQ ID NO: 89.

In other embodiments, the anti-FcRH5 antibody comprises a binding domaincomprising the following six HVRs: (a) an HVR-H1 comprising the aminoacid sequence of SEQ ID NO: 1, (b) an HVR-H2 comprising the amino acidsequence of SEQ ID NO: 7, (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 9, (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 12, (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 16, and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 23. In some embodiments, the binding domaincomprises (a) a VH domain comprising an amino acid sequence having atleast 95% sequence identity to the amino acid sequence of SEQ ID NO: 90,(b) a VL domain comprising an amino acid sequence having at least 95%sequence identity to the amino acid sequence of SEQ ID NO: 91, or (c) aVH domain as in (a) and a VL domain as in (b). In some embodiments, theantibody further comprises the following heavy chain variable regionFRs: (a) an FR-H1 comprising the amino acid sequence of SEQ ID NO: 52,(b) an FR-H2 comprising the amino acid sequence of SEQ ID NO: 54, (c) anFR-H3 comprising the amino acid sequence of SEQ ID NO: 46, and (d) anFR-H4 comprising the amino acid sequence of SEQ ID NO: 47. In someembodiments, the VH domain comprises the amino acid sequence of SEQ IDNO: 90. In some embodiments, the antibody further comprises thefollowing light chain variable region FRs: (a) an FR-L1 comprising theamino acid sequence of SEQ ID NO: 48, (b) an FR-L2 comprising the aminoacid sequence of SEQ ID NO: 57, (c) an FR-L3 comprising the amino acidsequence of SEQ ID NO: 50, and (d) an FR-L4 comprising the amino acidsequence of SEQ ID NO: 51. In some embodiments, the VL domain comprisesthe amino acid sequence of SEQ ID NO: 91. In some embodiments, theanti-FcRH5 antibody comprises a binding domain comprising (a) a VHdomain comprising an amino acid sequence of SEQ ID NO: 90 and (b) a VLdomain comprising an amino acid sequence of SEQ ID NO: 91.

In other embodiments, the anti-FcRH5 antibody comprises a binding domaincomprising the following six HVRs: (a) an HVR-H1 comprising the aminoacid sequence of SEQ ID NO: 1, (b) an HVR-H2 comprising the amino acidsequence of SEQ ID NO: 7, (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 9, (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 11, (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 18, and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 22. In some embodiments, the binding domaincomprises (a) a VH domain comprising an amino acid sequence having atleast 95% sequence identity to the amino acid sequence of SEQ ID NO: 92,(b) a VL domain comprising an amino acid sequence having at least 95%sequence identity to the amino acid sequence of SEQ ID NO: 93, or (c) aVH domain as in (a) and a VL domain as in (b). In some embodiments, theantibody further comprises the following heavy chain variable regionFRs: (a) an FR-H1 comprising the amino acid sequence of SEQ ID NO: 52,(b) an FR-H2 comprising the amino acid sequence of SEQ ID NO: 54, (c) anFR-H3 comprising the amino acid sequence of SEQ ID NO: 46, and (d) anFR-H4 comprising the amino acid sequence of SEQ ID NO: 47. In someembodiments, the VH domain comprises the amino acid sequence of SEQ IDNO: 92. In some embodiments, the antibody further comprises thefollowing light chain variable region FRs: (a) an FR-L1 comprising theamino acid sequence of SEQ ID NO: 48, (b) an FR-L2 comprising the aminoacid sequence of SEQ ID NO: 56, (c) an FR-L3 comprising the amino acidsequence of SEQ ID NO: 50, and (d) an FR-L4 comprising the amino acidsequence of SEQ ID NO: 51. In some embodiments, the VL domain comprisesthe amino acid sequence of SEQ ID NO: 93. In some embodiments, theanti-FcRH5 antibody comprises a binding domain comprising (a) a VHdomain comprising an amino acid sequence of SEQ ID NO: 92 and (b) a VLdomain comprising an amino acid sequence of SEQ ID NO: 93.

In other embodiments, the anti-FcRH5 antibody comprises a binding domaincomprising the following six HVRs: (a) an HVR-H1 comprising the aminoacid sequence of SEQ ID NO: 1, (b) an HVR-H2 comprising the amino acidsequence of SEQ ID NO: 7, (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 9, (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 11, (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 19, and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 24. In some embodiments, the binding domaincomprises (a) a VH domain comprising an amino acid sequence having atleast 95% sequence identity to the amino acid sequence of SEQ ID NO: 94,(b) a VL domain comprising an amino acid sequence having at least 95%sequence identity to the amino acid sequence of SEQ ID NO: 95, or (c) aVH domain as in (a) and a VL domain as in (b). In some embodiments, theantibody further comprises the following heavy chain variable regionFRs: (a) an FR-H1 comprising the amino acid sequence of SEQ ID NO: 52,(b) an FR-H2 comprising the amino acid sequence of SEQ ID NO: 54, (c) anFR-H3 comprising the amino acid sequence of SEQ ID NO: 46, and (d) anFR-H4 comprising the amino acid sequence of SEQ ID NO: 47. In someembodiments, the VH domain comprises the amino acid sequence of SEQ IDNO: 94. In some embodiments, the antibody further comprises thefollowing light chain variable region FRs: (a) an FR-L1 comprising theamino acid sequence of SEQ ID NO: 48, (b) an FR-L2 comprising the aminoacid sequence of SEQ ID NO: 57, (c) an FR-L3 comprising the amino acidsequence of SEQ ID NO: 50, and (d) an FR-L4 comprising the amino acidsequence of SEQ ID NO: 51. In some embodiments, the VL domain comprisesthe amino acid sequence of SEQ ID NO: 95. In some embodiments, theanti-FcRH5 antibody comprises a binding domain comprising (a) a VHdomain comprising an amino acid sequence of SEQ ID NO: 94 and (b) a VLdomain comprising an amino acid sequence of SEQ ID NO: 95.

In other embodiments, the anti-FcRH5 antibody comprises a binding domaincomprising the following six HVRs: (a) an HVR-H1 comprising the aminoacid sequence of SEQ ID NO: 1, (b) an HVR-H2 comprising the amino acidsequence of SEQ ID NO: 7, (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 9, (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 12, (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 18, and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 25. In some embodiments, the binding domaincomprises (a) a VH domain comprising an amino acid sequence having atleast 95% sequence identity to the amino acid sequence of SEQ ID NO: 96,(b) a VL domain comprising an amino acid sequence having at least 95%sequence identity to the amino acid sequence of SEQ ID NO: 97, or (c) aVH domain as in (a) and a VL domain as in (b). In some embodiments, theantibody further comprises the following heavy chain variable regionFRs: (a) an FR-H1 comprising the amino acid sequence of SEQ ID NO: 53,(b) an FR-H2 comprising the amino acid sequence of SEQ ID NO: 54, (c) anFR-H3 comprising the amino acid sequence of SEQ ID NO: 46, and (d) anFR-H4 comprising the amino acid sequence of SEQ ID NO: 47. In someembodiments, the VH domain comprises the amino acid sequence of SEQ IDNO: 96. In some embodiments, the antibody further comprises thefollowing light chain variable region FRs: (a) an FR-L1 comprising theamino acid sequence of SEQ ID NO: 48, (b) an FR-L2 comprising the aminoacid sequence of SEQ ID NO: 57, (c) an FR-L3 comprising the amino acidsequence of SEQ ID NO: 50, and (d) an FR-L4 comprising the amino acidsequence of SEQ ID NO: 51. In some embodiments, the VL domain comprisesthe amino acid sequence of SEQ ID NO: 97. In some embodiments, theanti-FcRH5 antibody comprises a binding domain comprising (a) a VHdomain comprising an amino acid sequence of SEQ ID NO: 96 and (b) a VLdomain comprising an amino acid sequence of SEQ ID NO: 97.

In other embodiments, the anti-FcRH5 antibody comprises a binding domaincomprising the following six HVRs: (a) an HVR-H1 comprising the aminoacid sequence of SEQ ID NO: 1, (b) an HVR-H2 comprising the amino acidsequence of SEQ ID NO: 7, (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 9, (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 12, (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 18, and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 25. In some embodiments, the binding domaincomprises (a) a VH domain comprising an amino acid sequence having atleast 95% sequence identity to the amino acid sequence of SEQ ID NO: 98,(b) a VL domain comprising an amino acid sequence having at least 95%sequence identity to the amino acid sequence of SEQ ID NO: 99, or (c) aVH domain as in (a) and a VL domain as in (b). In some embodiments, theantibody further comprises the following heavy chain variable regionFRs: (a) an FR-H1 comprising the amino acid sequence of SEQ ID NO: 52,(b) an FR-H2 comprising the amino acid sequence of SEQ ID NO: 55, (c) anFR-H3 comprising the amino acid sequence of SEQ ID NO: 46, and (d) anFR-H4 comprising the amino acid sequence of SEQ ID NO: 47. In someembodiments, the VH domain comprises the amino acid sequence of SEQ IDNO: 98. In some embodiments, the antibody further comprises thefollowing light chain variable region FRs: (a) an FR-L1 comprising theamino acid sequence of SEQ ID NO: 48, (b) an FR-L2 comprising the aminoacid sequence of SEQ ID NO: 57, (c) an FR-L3 comprising the amino acidsequence of SEQ ID NO: 50, and (d) an FR-L4 comprising the amino acidsequence of SEQ ID NO: 51. In some embodiments, the VL domain comprisesthe amino acid sequence of SEQ ID NO: 99. In some embodiments, theanti-FcRH5 antibody comprises a binding domain comprising (a) a VHdomain comprising an amino acid sequence of SEQ ID NO: 98 and (b) a VLdomain comprising an amino acid sequence of SEQ ID NO: 99.

In other embodiments, the anti-FcRH5 antibody comprises a binding domaincomprising the following six HVRs: (a) an HVR-H1 comprising the aminoacid sequence of SEQ ID NO: 1, (b) an HVR-H2 comprising the amino acidsequence of SEQ ID NO: 7, (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 9, (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 12, (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 18, and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 25. In some embodiments, the binding domaincomprises (a) a VH domain comprising an amino acid sequence having atleast 95% sequence identity to the amino acid sequence of SEQ ID NO:100, (b) a VL domain comprising an amino acid sequence having at least95% sequence identity to the amino acid sequence of SEQ ID NO: 101, or(c) a VH domain as in (a) and a VL domain as in (b). In someembodiments, the antibody further comprises the following heavy chainvariable region FRs: (a) an FR-H1 comprising the amino acid sequence ofSEQ ID NO: 52, (b) an FR-H2 comprising the amino acid sequence of SEQ IDNO: 54, (c) an FR-H3 comprising the amino acid sequence of SEQ ID NO:46, and (d) an FR-H4 comprising the amino acid sequence of SEQ ID NO:47. In some embodiments, the VH domain comprises the amino acid sequenceof SEQ ID NO: 100. In some embodiments, the antibody further comprisesthe following light chain variable region FRs: (a) an FR-L1 comprisingthe amino acid sequence of SEQ ID NO: 48, (b) an FR-L2 comprising theamino acid sequence of SEQ ID NO: 57, (c) an FR-L3 comprising the aminoacid sequence of SEQ ID NO: 50, and (d) an FR-L4 comprising the aminoacid sequence of SEQ ID NO: 51. In some embodiments, the VL domaincomprises the amino acid sequence of SEQ ID NO: 101. In someembodiments, the anti-FcRH5 antibody comprises a binding domaincomprising (a) a VH domain comprising an amino acid sequence of SEQ IDNO: 100 and (b) a VL domain comprising an amino acid sequence of SEQ IDNO: 101.

In other embodiments, the anti-FcRH5 antibody comprises a binding domaincomprising the following six HVRs: (a) an HVR-H1 comprising the aminoacid sequence of SEQ ID NO: 1, (b) an HVR-H2 comprising the amino acidsequence of SEQ ID NO: 8, (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 9, (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 11, (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 15, and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 20. In some embodiments, the binding domaincomprises (a) a VH domain comprising an amino acid sequence having atleast 95% sequence identity to the amino acid sequence of SEQ ID NO:102, (b) a VL domain comprising an amino acid sequence having at least95% sequence identity to the amino acid sequence of SEQ ID NO: 103, or(c) a VH domain as in (a) and a VL domain as in (b). In someembodiments, the antibody further comprises the following heavy chainvariable region FRs: (a) an FR-H1 comprising the amino acid sequence ofSEQ ID NO: 52, (b) an FR-H2 comprising the amino acid sequence of SEQ IDNO: 54, (c) an FR-H3 comprising the amino acid sequence of SEQ ID NO:46, and (d) an FR-H4 comprising the amino acid sequence of SEQ ID NO:47. In some embodiments, the VH domain comprises the amino acid sequenceof SEQ ID NO: 102. In some embodiments, the antibody further comprisesthe following light chain variable region FRs: (a) an FR-L1 comprisingthe amino acid sequence of SEQ ID NO: 48, (b) an FR-L2 comprising theamino acid sequence of SEQ ID NO: 56, (c) an FR-L3 comprising the aminoacid sequence of SEQ ID NO: 50, and (d) an FR-L4 comprising the aminoacid sequence of SEQ ID NO: 51. In some embodiments, the VL domaincomprises the amino acid sequence of SEQ ID NO: 103. In someembodiments, the anti-FcRH5 antibody comprises a binding domaincomprising (a) a VH domain comprising an amino acid sequence of SEQ IDNO: 102 and (b) a VL domain comprising an amino acid sequence of SEQ IDNO: 103.

In another aspect, the invention features an anti-FcRH5 antibodyincluding a binding domain comprising the following six HVRs: (a) anHVR-H1 comprising the amino acid sequence of SEQ ID NO: 32, (b) anHVR-H2 comprising the amino acid sequence of SEQ ID NO: 33, (c) anHVR-H3 comprising the amino acid sequence of SEQ ID NO: 34, (d) anHVR-L1 comprising the amino acid sequence of SEQ ID NO: 35, (e) anHVR-L2 comprising the amino acid sequence of SEQ ID NO: 36, and (f) anHVR-L3 comprising the amino acid sequence of SEQ ID NO: 37. In someembodiments, the binding domain comprises (a) a VH domain comprising anamino acid sequence having at least 95% sequence identity to the aminoacid sequence of SEQ ID NO: 110, (b) a VL domain comprising an aminoacid sequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO: 111, or (c) a VH domain as in (a) and a VL domainas in (b). In some embodiments, the antibody further comprises thefollowing heavy chain variable region FRs: (a) an FR-H1 comprising theamino acid sequence of SEQ ID NO: 66, (b) an FR-H2 comprising the aminoacid sequence of SEQ ID NO: 67, (c) an FR-H3 comprising the amino acidsequence of SEQ ID NO: 68, and (d) an FR-H4 comprising the amino acidsequence of SEQ ID NO: 69. In some embodiments, the VH domain comprisesthe amino acid sequence of SEQ ID NO: 110. In some embodiments, theantibody further comprises the following light chain variable regionFRs: (a) an FR-L1 comprising the amino acid sequence of SEQ ID NO: 70,(b) an FR-L2 comprising the amino acid sequence of SEQ ID NO: 71, (c) anFR-L3 comprising the amino acid sequence of SEQ ID NO: 72, and (d) anFR-L4 comprising the amino acid sequence of SEQ ID NO: 73. In someembodiments, the VL domain comprises the amino acid sequence of SEQ IDNO: 111. In some embodiments, the anti-FcRH5 antibody comprises abinding domain comprising (a) a VH domain comprising an amino acidsequence of SEQ ID NO: 110 and (b) a VL domain comprising an amino acidsequence of SEQ ID NO: 111.

In another aspect, the invention features an anti-FcRH5 antibodyincluding a binding domain comprising the following six HVRs: (a) anHVR-H1 comprising the amino acid sequence of SEQ ID NO: 38, (b) anHVR-H2 comprising the amino acid sequence of SEQ ID NO: 39, (c) anHVR-H3 comprising the amino acid sequence of SEQ ID NO: 40, (d) anHVR-L1 comprising the amino acid sequence of SEQ ID NO: 41, (e) anHVR-L2 comprising the amino acid sequence of SEQ ID NO: 42, and (f) anHVR-L3 comprising the amino acid sequence of SEQ ID NO: 43. In otherembodiments, the binding domain comprises (a) a VH domain comprising anamino acid sequence having at least 95% sequence identity to the aminoacid sequence of SEQ ID NO: 112, (b) a VL domain comprising an aminoacid sequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO: 113, or (c) a VH domain as in (a) and a VL domainas in (b). In other embodiments, the antibody further comprises thefollowing heavy chain variable region FRs: (a) an FR-H1 comprising theamino acid sequence of SEQ ID NO: 74, (b) an FR-H2 comprising the aminoacid sequence of SEQ ID NO: 75, (c) an FR-H3 comprising the amino acidsequence of SEQ ID NO: 76, and (d) an FR-H4 comprising the amino acidsequence of SEQ ID NO: 77. In other embodiments, the VH domain comprisesthe amino acid sequence of SEQ ID NO: 112. In other embodiments, theantibody further comprises the following light chain variable regionFRs: (a) an FR-L1 comprising the amino acid sequence of SEQ ID NO: 78,(b) an FR-L2 comprising the amino acid sequence of SEQ ID NO: 79, (c) anFR-L3 comprising the amino acid sequence of SEQ ID NO: 80, and (d) anFR-L4 comprising the amino acid sequence of SEQ ID NO: 81. In otherembodiments, the VL domain comprises the amino acid sequence of SEQ IDNO: 113. In other embodiments, the anti-FcRH5 antibody comprises abinding domain comprising (a) a VH domain comprising an amino acidsequence of SEQ ID NO: 112 and (b) a VL domain comprising an amino acidsequence of SEQ ID NO: 113.

In another aspect, the invention features an anti-FcRH5 antibodyincluding a binding domain comprising the following six HVRs: (a) anHVR-H1 comprising the amino acid sequence of SEQ ID NO: 26, (b) anHVR-H2 comprising the amino acid sequence of SEQ ID NO: 27, (c) anHVR-H3 comprising the amino acid sequence of SEQ ID NO: 28, (d) anHVR-L1 comprising the amino acid sequence of SEQ ID NO: 29, (e) anHVR-L2 comprising the amino acid sequence of SEQ ID NO: 30, and (f) anHVR-L3 comprising the amino acid sequence of SEQ ID NO: 31. In someembodiments, the binding domain comprises (a) a VH domain comprising anamino acid sequence having at least 95% sequence identity to the aminoacid sequence of SEQ ID NO: 108, (b) a VL domain comprising an aminoacid sequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO: 109, or (c) a VH domain as in (a) and a VL domainas in (b). In some embodiments, the antibody further comprises thefollowing heavy chain variable region FRs: (a) an FR-H1 comprising theamino acid sequence of SEQ ID NO: 58, (b) an FR-H2 comprising the aminoacid sequence of SEQ ID NO: 59, (c) an FR-H3 comprising the amino acidsequence of SEQ ID NO: 60, and (d) an FR-H4 comprising the amino acidsequence of SEQ ID NO: 61. In some embodiments, the VH domain comprisesthe amino acid sequence of SEQ ID NO: 108. In some embodiments, theantibody further comprises the following light chain variable regionFRs: (a) an FR-L1 comprising the amino acid sequence of SEQ ID NO: 62,(b) an FR-L2 comprising the amino acid sequence of SEQ ID NO: 63, (c) anFR-L3 comprising the amino acid sequence of SEQ ID NO: 64, and (d) anFR-L4 comprising the amino acid sequence of SEQ ID NO: 65. In someembodiments, the VL domain comprises the amino acid sequence of SEQ IDNO: 109. In some embodiments, the anti-FcRH5 antibody comprises abinding domain comprising (a) a VH domain comprising an amino acidsequence of SEQ ID NO: 108 and (b) a VL domain comprising an amino acidsequence of SEQ ID NO: 109.

In some embodiments of any one of the preceding aspects, the anti-FcRH5antibody binds to an epitope in the Ig-like domain 9 of FcRH5. In someembodiments, the epitope comprises a portion of amino acids 743-850 ofSEQ ID NO: 114. In some embodiments, the binding domain binds to humanFcRH5, cynomolgus monkey (cyno) FcRH5, or both. In some embodiments, thebinding domain does not specifically bind to FcRH1, FcRH2, FcRH3, and/orFcRH4. In some embodiments, the anti-FcRH5 antibody binds human FcRH5with a K_(D) of about 100 nM or lower. In some embodiments, theanti-FcRH5 antibody binds human FcRH5 with a K_(D) of between about 10pM and about 100 nM. In some embodiments, the anti-FcRH5 antibody bindshuman FcRH5 with a K_(D) of between about 100 pM and about 100 nM. Insome embodiments, the anti-FcRH5 antibody binds human FcRH5 with a K_(D)of between about 1 nM and about 20 nM. In some embodiments, theanti-FcRH5 antibody binds human FcRH5 with a K_(D) of between about 1 nMand about 10 nM. In some embodiments, the anti-FcRH5 antibody binds cynoFcRH5 with a K_(D) of about 100 nM or lower. In some embodiments, theanti-FcRH5 antibody binds cyno FcRH5 with a K_(D) of between about 10 pMand about 100 nM. In some embodiments, the anti-FcRH5 antibody bindscyno FcRH5 with a K_(D) of between about 100 pM and about 100 nM. Insome embodiments, the anti-FcRH5 antibody binds cyno FcRH5 with a K_(D)of between about 1 nM and about 50 nM.

In other embodiments, the anti-FcRH5 antibody comprises anaglycosylation site mutation. In some embodiments, the aglycosylationsite mutation is a substitution mutation. In some embodiments, theaglycosylation site mutation reduces effector function of the anti-FcRH5antibody. In some embodiments, the substitution mutation is at aminoacid residue N297, L234, L235, D265, and/or P329 (EU numbering). In someembodiments, the substitution mutation is selected from the groupconsisting of N297G, N297A, L234A, L235A, D265A, and P329G. In someembodiments, the substitution mutation is an N297G mutation.

In other embodiments, the anti-FcRH5 antibody is an IgG antibody.

In some embodiments, the anti-FcRH5 antibody is an antibody fragmentthat binds FcRH5. In some embodiments, the antibody fragment is selectedfrom the group consisting of bis-Fab, Fab, Fab′-SH, Fv, scFv, and(Fab′)₂ fragments. In some embodiments, the antibody fragment is abis-Fab fragment.

In other embodiments, the anti-FcRH5 antibody is a full-length antibody.

In some embodiments, the anti-FcRH5 antibody is a monospecific antibody.

In some embodiments, the anti-FcRH5 antibody is a multispecificantibody. In some embodiments, the multispecific antibody is abispecific antibody. In some embodiments, the bispecific antibodycomprises a second binding domain that binds cluster of differentiation3 (CD3). In some embodiments, the second binding domain binds to anepitope on CD3 comprising amino acid residue Glu6 of CD3. In someembodiments, the epitope further comprises one or more additional aminoacid residues selected from the group consisting of Gln1, Asp2, and Met7of CD3. In some embodiments, the epitope comprises amino acid residuesGln1, Asp2, and Glu6 of CD3. In some embodiments, the epitope comprisesamino acid residues Gln1, Asp2, Glu6, and Met7 of CD3. In someembodiments, the epitope does not comprise amino acid residue Glu5 ofCD3. In some embodiments, the epitope does not comprise amino acidresidues Gly3 and Glu5 of CD3. In some embodiments, the epitope consistsof amino acid residues Gln1, Asp2, Glu6, and Met7 of CD3. In someembodiments, the second binding domain is capable of binding to a humanCD3 polypeptide or a cyno CD3 polypeptide. In some embodiments, thehuman CD3 polypeptide or the cyno CD3 polypeptide is a human CD3εpolypeptide or a cyno CD3ε polypeptide, respectively. In someembodiments, the human CD3 polypeptide or the cyno CD3 polypeptide is ahuman CD3γ polypeptide or a cyno CD3γ polypeptide, respectively. In someembodiments, the second binding domain binds the human CD3ε polypeptidewith a K_(D) of about 100 nM or lower. In some embodiments, the secondbinding domain binds the human CD3ε polypeptide with a K_(D) of betweenabout 10 pM to about 100 nM. In some embodiments, the second bindingdomain binds the human CD3ε polypeptide with a K_(D) of between about100 pM to about 50 nM. In some embodiments, the second binding domainbinds the human CD3ε polypeptide with a K_(D) of between about 1 nM toabout 10 nM.

In some embodiments, the second binding domain comprises the followingsix HVRs: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:115, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 116,(c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 117, (d)an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 118, (e) anHVR-L2 comprising the amino acid sequence of SEQ ID NO: 119, and (f) anHVR-L3 comprising the amino acid sequence of SEQ ID NO: 120. In someembodiments, the second binding domain comprises the following six HVRs:(a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 115, (b)an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 116, (c) anHVR-H3 comprising the amino acid sequence of SEQ ID NO: 121, (d) anHVR-L1 comprising the amino acid sequence of SEQ ID NO: 118, (e) anHVR-L2 comprising the amino acid sequence of SEQ ID NO: 119, and (f) anHVR-L3 comprising the amino acid sequence of SEQ ID NO: 123. In someembodiments, the second binding domain comprises (a) a VH domaincomprising an amino acid sequence having at least 95% sequence identityto the amino acid sequence of SEQ ID NO: 133, (b) a VL domain comprisingan amino acid sequence having at least 95% sequence identity to theamino acid sequence of SEQ ID NO: 134, or (c) a VH domain as in (a) anda VL domain as in (b). In some embodiments, the second binding domaincomprises the following heavy chain variable region FRs: (a) an FR-H1comprising the amino acid sequence of SEQ ID NO: 125, (b) an FR-H2comprising the amino acid sequence of SEQ ID NO: 126, (c) an FR-H3comprising the amino acid sequence of SEQ ID NO: 127, and (d) an FR-H4comprising the amino acid sequence of SEQ ID NO: 128. In someembodiments, the second binding domain comprises a VH domain comprisingthe amino acid sequence of SEQ ID NO: 133. In some embodiments, thesecond binding domain further comprises the following light chainvariable region FRs: (a) an FR-L1 comprising the amino acid sequence ofSEQ ID NO: 129, (b) an FR-L2 comprising the amino acid sequence of SEQID NO: 130, (c) an FR-L3 comprising the amino acid sequence of SEQ IDNO: 131, and (d) an FR-L4 comprising the amino acid sequence of SEQ IDNO: 132. In some embodiments, the second binding domain comprises a VLdomain comprising the amino acid sequence of SEQ ID NO: 134. In someembodiments, the second binding domain comprises (a) a VH domaincomprising an amino acid sequence of SEQ ID NO: 133 and (b) a VL domaincomprising an amino acid sequence of SEQ ID NO: 134.

In other embodiments, the second binding domain comprises the followingsix HVRs: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:115, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 116,(c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121, (d)an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 118, (e) anHVR-L2 comprising the amino acid sequence of SEQ ID NO: 119, and (f) anHVR-L3 comprising the amino acid sequence of SEQ ID NO: 124. In someembodiments, the second binding domain comprises (a) a VH domaincomprising an amino acid sequence having at least 95% sequence identityto the amino acid sequence of SEQ ID NO: 137, (b) a VL domain comprisingan amino acid sequence having at least 95% sequence identity to theamino acid sequence of SEQ ID NO: 138, or (c) a VH domain as in (a) anda VL domain as in (b). In some embodiments, the second binding domaincomprises the following heavy chain variable region FRs: (a) an FR-H1comprising the amino acid sequence of SEQ ID NO: 125, (b) an FR-H2comprising the amino acid sequence of SEQ ID NO: 126, (c) an FR-H3comprising the amino acid sequence of SEQ ID NO: 127, and (d) an FR-H4comprising the amino acid sequence of SEQ ID NO: 128. In someembodiments, the second binding domain comprises a VH domain comprisingthe amino acid sequence of SEQ ID NO: 137. In some embodiments, thesecond binding domain further comprises the following light chainvariable region FRs: (a) an FR-L1 comprising the amino acid sequence ofSEQ ID NO: 129, (b) an FR-L2 comprising the amino acid sequence of SEQID NO: 130, (c) an FR-L3 comprising the amino acid sequence of SEQ IDNO: 131, and (d) an FR-L4 comprising the amino acid sequence of SEQ IDNO: 132. In some embodiments, the second binding domain comprises a VLdomain comprising the amino acid sequence of SEQ ID NO: 138. In someembodiments, the second binding domain comprises (a) a VH domaincomprising an amino acid sequence of SEQ ID NO: 137 and (b) a VL domaincomprising an amino acid sequence of SEQ ID NO: 138.

In other embodiments, the second binding domain comprises the followingsix HVRs: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:139, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 140,(c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 141, (d)an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 142, (e) anHVR-L2 comprising the amino acid sequence of SEQ ID NO: 143, and (f) anHVR-L3 comprising the amino acid sequence of SEQ ID NO: 144. In someembodiments, the second binding domain comprises (a) a VH domaincomprising an amino acid sequence having at least 95% sequence identityto the amino acid sequence of SEQ ID NO: 153, (b) a VL domain comprisingan amino acid sequence having at least 95% sequence identity to theamino acid sequence of SEQ ID NO: 154, or (c) a VH domain as in (a) anda VL domain as in (b). In some embodiments, the second binding domaincomprises the following heavy chain variable region FRs: (a) an FR-H1comprising the amino acid sequence of SEQ ID NO: 145, (b) an FR-H2comprising the amino acid sequence of SEQ ID NO: 146, (c) an FR-H3comprising the amino acid sequence of SEQ ID NO: 147, and (d) an FR-H4comprising the amino acid sequence of SEQ ID NO: 148. In someembodiments, the second binding domain comprises a VH domain comprisingthe amino acid sequence of SEQ ID NO: 153. In some embodiments, thesecond binding domain further comprises the following light chainvariable region FRs: (a) an FR-L1 comprising the amino acid sequence ofSEQ ID NO: 149, (b) an FR-L2 comprising the amino acid sequence of SEQID NO: 150, (c) an FR-L3 comprising the amino acid sequence of SEQ IDNO: 151, and (d) an FR-L4 comprising the amino acid sequence of SEQ IDNO: 152. In some embodiments, the second binding domain comprises a VLdomain comprising the amino acid sequence of SEQ ID NO: 154. In someembodiments, the second binding domain comprises (a) a VH domaincomprising an amino acid sequence of SEQ ID NO: 153 and (b) a VL domaincomprising an amino acid sequence of SEQ ID NO: 154.

In other embodiments, the second binding domain comprises the followingsix HVRs: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:155, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 156,(c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 157, (d)an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 158, (e) anHVR-L2 comprising the amino acid sequence of SEQ ID NO: 159, and (f) anHVR-L3 comprising the amino acid sequence of SEQ ID NO: 160.

In some embodiments, the second binding domain comprises the followingsix HVRs: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:155, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 162,(c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 157, (d)an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 158, (e) anHVR-L2 comprising the amino acid sequence of SEQ ID NO: 159, and (f) anHVR-L3 comprising the amino acid sequence of SEQ ID NO: 160. In someembodiments, the second binding domain comprises (a) a VH domaincomprising an amino acid sequence having at least 95% sequence identityto the amino acid sequence of SEQ ID NO: 172, (b) a VL domain comprisingan amino acid sequence having at least 95% sequence identity to theamino acid sequence of SEQ ID NO: 173, or (c) a VH domain as in (a) anda VL domain as in (b). In some embodiments, the second binding domaincomprises the following heavy chain variable region FRs: (a) an FR-H1comprising the amino acid sequence of SEQ ID NO: 164, (b) an FR-H2comprising the amino acid sequence of SEQ ID NO: 165, (c) an FR-H3comprising the amino acid sequence of SEQ ID NO: 166, and (d) an FR-H4comprising the amino acid sequence of SEQ ID NO: 167. In someembodiments, the second binding domain comprises a VH domain comprisingthe amino acid sequence of SEQ ID NO: 172. In some embodiments, thesecond binding domain further comprises the following light chainvariable region FRs: (a) an FR-L1 comprising the amino acid sequence ofSEQ ID NO: 168, (b) an FR-L2 comprising the amino acid sequence of SEQID NO: 169, (c) an FR-L3 comprising the amino acid sequence of SEQ IDNO: 170, and (d) an FR-L4 comprising the amino acid sequence of SEQ IDNO: 171. In some embodiments, the second binding domain comprises a VLdomain comprising the amino acid sequence of SEQ ID NO: 173. In someembodiments, the second binding domain comprises (a) a VH domaincomprising an amino acid sequence of SEQ ID NO: 172 and (b) a VL domaincomprising an amino acid sequence of SEQ ID NO: 173.

In other embodiments, the binding domain that binds FcRH5 comprises a VHdomain (VH₁) comprising a charged region (CR₁) and a VL domain (VL₁)comprising a charged region (CR₂), wherein the CR₁ in the VH₁ forms acharge pair with the CR₂ in the VL₁. In some embodiments, the CR₁comprises a basic amino acid residue and the CR₂ comprises an acidicamino acid residue. In some embodiments, the CR₁ comprises a Q39Ksubstitution mutation (EU numbering). In some embodiments, the CR₁consists of the Q39K substitution mutation. In some embodiments, the CR₂comprises a Q38E substitution mutation (EU numbering). In someembodiments, the CR₂ consists of the Q38E substitution mutation. In someembodiments, the second binding domain that binds CD3 comprises a VHdomain (VH₂) comprising a charged region (CR₃) and a VL domain (VL₂)comprising a charged region (CR₄), wherein the CR₄ in the VL₂ forms acharge pair with the CR₃ in the VH₂. In some embodiments, the CR₄comprises a basic amino acid residue and the CR₃ comprises an acidicamino acid residue. In some embodiments, the CR₄ comprises a Q38Ksubstitution mutation (EU numbering). In some embodiments, the CR₄consists of the Q38K substitution mutation. In some embodiments, the CR₃comprises a Q39E substitution mutation (EU numbering). In someembodiments, the CR₃ consists of the Q39E substitution mutation. In someembodiments, the VL₁ domain is linked to a light chain constant (CL)domain (CL₁) and the VH₁ is linked to a first heavy chain constant (CH1)domain (CH1₁), wherein the CL₁ comprises a charged region (CR₅) and theCH1₁ comprises a charged region (CR₆), and wherein the CR₅ in the CL₁forms a charge pair with the CR₆ in the CH1₁. In some embodiments, theCR₅ comprises a basic amino acid residue and the CR₆ comprises an acidicresidue. In some embodiments, the CR₅ comprises a V133K substitutionmutation (EU numbering). In some embodiments, the CR₅ consists of theV133K substitution mutation. In some embodiments, the CR₆ comprises aS183E substitution mutation (EU numbering). In some embodiments, the CR₆consists of the S183E substitution mutation.

In other embodiments, the VL₂ domain is linked to a CL domain (CL₂) andthe VH₂ is linked to a CH1 domain (CH1₂), wherein the CL₂ comprises acharged region (CR₇) and the CH1₂ comprise a charged region (CR₈), andwherein the CR₈ in the CH1₂ forms a charge pair with the CR₇ in the CL₂.In some embodiments, the CR₈ comprises a basic amino acid residue andthe CR₇ comprises an acidic amino acid residue. In some embodiments, theCR₈ comprises a S183K substitution mutation (EU numbering). In someembodiments, the CR₈ consists of the S183K substitution mutation. Insome embodiments, the CR₇ comprises a V133E substitution mutation (EUnumbering). In some embodiments, the CR₇ consists of the V133Esubstitution mutation.

In other embodiments, the VL₂ domain is linked to a CL domain (CL₂) andthe VH₂ is linked to a CH1 domain (CH1₂), wherein (a) the CL₂ comprisesone or more mutations at amino acid residues F116, L135, S174, S176,and/or T178 (EU numbering) and (b) the CH1₂ comprises one or moremutations at amino acid residues A141, F170, S181, S183, and/or V185 (EUnumbering). In some embodiments, the CL₂ comprises one or more of thefollowing substitution mutations: F116A, L135V, S174A, S176F, and/orT178V. In some embodiments, the CL₂ comprises the following substitutionmutations: F116A, L135V, S174A, S176F, and T178V. In some embodiments,the CH1₂ comprises one or more of the following substitution mutations:A141I, F170S, S181M, S183A, and/or V185A. In some embodiments, the CH1₂comprises the following substitution mutations: A141I, F170S, S181M,S183A, and V185A.

In other embodiments, the binding domain that binds FcRH5 comprises a VHdomain (VH₁) comprising a charged region (CR₁) and a VL domain (VL₁)comprising a charged region (CR₂), wherein the CR₂ in the VL₁ forms acharge pair with the CR₁ in the VH₁. In some embodiments, the CR₂comprises a basic amino acid residue and the CR₁ comprises an acidicamino acid residue. In some embodiments, the CR₂ comprises a Q38Ksubstitution mutation (EU numbering). In some embodiments, the CR₂consists of the Q38K substitution mutation. In some embodiments, the CR₁comprises a Q39E substitution mutation (EU numbering). In someembodiments, the CR₁ consists of the Q39E substitution mutation. In someembodiments, the second binding domain that binds CD3 comprises a VHdomain (VH₂) comprising a charged region (CR₃) and a VL domain (VL₂)comprising a charged region (CR₄), wherein the CR₃ in the VH₂ forms acharge pair with the CR₄ in the VL₂. In some embodiments, the CR₃comprises a basic amino acid residue and the CR₄ comprises an acidicamino acid residue. In some embodiments, the CR₃ comprises a Q39Ksubstitution mutation (EU numbering). In some embodiments, the CR₃consists of the Q39K substitution mutation. In some embodiments, the CR₄comprises a Q38E substitution mutation (EU numbering). In someembodiments, the CR₄ consists of the Q38E substitution mutation. In someembodiments, the VL₁ domain is linked to a light chain constant (CL)domain (CL₁) and the VH₁ is linked to a first heavy chain constant (CH1)domain (CH1₁), wherein the CL₁ comprises a charged region (CR₅) and theCH1₁ comprises a charged region (CR₆), and wherein the CR₆ in the CH1₁forms a charge pair with the CR₅ in the CL₁. In some embodiments, theCR₆ comprises a basic amino acid residue and the CR₅ comprises an acidicamino acid residue. In some embodiments, the CR₆ comprises a S183Ksubstitution mutation (EU numbering). In some embodiments, the CR₆consists of the S183K substitution mutation. In some embodiments, theCR₅ comprises a V133E substitution mutation (EU numbering). In someembodiments, the CR₅ consists of the V133E substitution mutation.

In other embodiments, the VL₂ domain is linked to a CL domain (CL₂) andthe VH₂ is linked to a CH1 domain (CH1₂), wherein the CL₂ comprises acharged region (CR₇) and the CH1₂ comprises a charged region (CR₈), andwherein the CR₇ in the CL₂ forms a charged pair with the CR₈ in theCH1₂. In some embodiments, the CR₇ comprises a basic amino acid residueand the CR₈ comprises an acidic residue. In some embodiments, the CR₇comprises a V133K substitution mutation (EU numbering). In someembodiments, the CR₇ consists of the V133K substitution mutation. Insome embodiments, the CR₈ comprises a S183E substitution mutation (EUnumbering). In some embodiments, the CR₈ consists of the S183Esubstitution mutation.

In other embodiments, the VL₂ domain is linked to a CL domain (CL₂) andthe VH₂ is linked to a CH1 domain (CH1₂), wherein (a) the CL₂ comprisesone or more mutations at amino acid residues F116, L135, S174, S176,and/or T178 (EU numbering) and (b) the CH1₂ comprises one or moremutations at amino acid residues A141, F170, S181, S183, and/or V185 (EUnumbering). In some embodiments, the CL₂ comprises one or more of thefollowing substitution mutations: F116A, L135V, S174A, S176F, and/orT178V. In some embodiments, the CL₂ comprises the following substitutionmutations: F116A, L135V, S174A, S176F, and T178V. In some embodiments,the CH1₂ comprises one or more of the following substitution mutations:A141I, F170S, S181M, S183A, and/or V185A. In some embodiments, the CH1₂comprises the following substitution mutations: A141I, F170S, S181M,S183A, and V185A. In some embodiments, the anti-FcRH5 antibody comprisesone or more heavy chain constant domains, wherein the one or more heavychain constant domains are selected from a first CH2 domain (CH2₁), afirst CH3 domain (CH3₁), a second CH2 domain (CH2₂), and a second CH3domain (CH3₂). In some embodiments, at least one of the one or moreheavy chain constant domains is paired with another heavy chain constantdomain. In some embodiments, the CH3₁ and the CH3₂ each comprise aprotuberance (P₁) or a cavity (C₁), and wherein the P₁ or the C₁ in theCH3₁ is positionable in the C₁ or the P₁, respectively, in the CH3₂. Insome embodiments, the CH3₁ and the CH3₂ meet at an interface between theP₁ and the C₁. In some embodiments, the CH2₁ and the CH2₂ each comprise(P₂) or a cavity (C₂), and wherein the P₂ or the C₂ in the CH2₁ ispositionable in the C₂ or the P₂, respectively, in the CH2₂. In someembodiments, the CH2₁ and the CH2₂ meet at an interface between the P₂and the C₂.

In another aspect, the invention features an anti-FcRH5 antibody thatbinds to FcRH5 and CD3, wherein the anti-FcRH5 antibody comprises ananti-FcRH5 arm comprising a first binding domain comprising thefollowing six HVRs: (a) an HVR-H1 comprising the amino acid sequence ofSEQ ID NO: 1, (b) an HVR-H2 comprising the amino acid sequence of SEQ IDNO: 8, (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 9,(d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12, (e)an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (f)an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23, and ananti-CD3 arm comprising a second binding domain comprising the followingsix HVRs: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:115, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 116,(c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121, (d)an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 118, (e) anHVR-L2 comprising the amino acid sequence of SEQ ID NO: 119, and (f) anHVR-L3 comprising the amino acid sequence of SEQ ID NO: 123, and whereinthe anti-FcRH5 arm and the anti-CD3 arm each comprise an N297Gsubstitution mutation (EU numbering), and wherein the anti-FcRH5 armcomprises a T366W substitution mutation and the anti-CD3 arm comprisesT366S, L368A, and Y407V substitution mutations.

In another aspect, the invention features an anti-FcRH5 antibody thatbinds to FcRH5 and CD3, wherein the anti-FcRH5 antibody comprises ananti-FcRH5 arm comprising a first binding domain comprising thefollowing six HVRs: (a) an HVR-H1 comprising the amino acid sequence ofSEQ ID NO: 1, (b) an HVR-H2 comprising the amino acid sequence of SEQ IDNO: 8, (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 9,(d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12, (e)an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (f)an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23, and ananti-CD3 arm comprising a second binding domain comprising the followingsix HVRs: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:115, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 116,(c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121, (d)an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 118, (e) anHVR-L2 comprising the amino acid sequence of SEQ ID NO: 119, and (f) anHVR-L3 comprising the amino acid sequence of SEQ ID NO: 123, and whereinthe anti-FcRH5 arm comprises a light chain comprising Q38E and V133Ksubstitution mutations and a heavy chain comprising Q39K, S183E, andN297G substitution mutations, and wherein the anti-CD3 arm comprises alight chain comprising Q38K and V133E substitution mutations and a heavychain comprising Q39E, S183K, and N297G substitution mutations (EUnumbering).

In another aspect, the invention features an anti-FcRH5 antibody thatbinds to FcRH5 and CD3, wherein the anti-FcRH5 antibody comprises: (a)an anti-FcRH5 arm comprising a first binding domain comprising a VHdomain comprising an amino acid sequence of SEQ ID NO: 104 and a VLdomain comprising an amino acid sequence of SEQ ID NO: 105, wherein theanti-FcRH5 arm comprises a light chain comprising Q38E and V133Ksubstitution mutations and a heavy chain comprising Q39K, S183E, andN297G substitution mutations, and (b) an anti-CD3 arm comprising asecond binding domain comprising a VH domain comprising an amino acidsequence of SEQ ID NO: 133 and a VL domain comprising an amino acidsequence of SEQ ID NO: 134, wherein the anti-CD3 arm comprises a lightchain comprising Q38K and V133E substitution mutations and a heavy chaincomprising Q39E, S183K, and N297G substitution mutations (EU numbering).

In another aspect, the invention features an anti-FcRH5 antibody thatbinds to FcRH5 and CD3, wherein the anti-FcRH5 antibody comprises ananti-FcRH5 arm comprising a first binding domain comprising thefollowing six HVRs: (a) an HVR-H1 comprising the amino acid sequence ofSEQ ID NO: 1, (b) an HVR-H2 comprising the amino acid sequence of SEQ IDNO: 8, (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 9,(d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12, (e)an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (f)an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23, and ananti-CD3 arm comprising a second binding domain comprising the followingsix HVRs: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:115, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 116,(c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121, (d)an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 118, (e) anHVR-L2 comprising the amino acid sequence of SEQ ID NO: 119, and (f) anHVR-L3 comprising the amino acid sequence of SEQ ID NO: 123, and whereinthe anti-FcRH5 arm comprises a light chain comprising Q38K and V133Esubstitution mutations and a heavy chain comprising Q39E, S183K, andN297G substitution mutations, and wherein the anti-CD3 arm comprises alight chain comprising Q38E and V133K substitution mutations and a heavychain comprising Q39K, S183E, and N297G substitution mutations (EUnumbering).

In another aspect, the invention features an anti-FcRH5 antibody thatbinds to FcRH5 and CD3, wherein the anti-FcRH5 antibody comprises: (a)an anti-FcRH5 arm comprising a first binding domain comprising a VHdomain comprising an amino acid sequence of SEQ ID NO: 104 and a VLdomain comprising an amino acid sequence of SEQ ID NO: 105, wherein theanti-FcRH5 arm comprises a light chain comprising Q38K and V133Esubstitution mutations and a heavy chain comprising Q39E, S183K, andN297G substitution mutations, and (b) an anti-CD3 arm comprising asecond binding domain comprising a VH domain comprising an amino acidsequence of SEQ ID NO: 133 and a VL domain comprising an amino acidsequence of SEQ ID NO: 134, wherein the anti-CD3 arm comprises a lightchain comprising Q38E and V133K substitution mutations and a heavy chaincomprising Q39K, S183E, and N297G substitution mutations (EU numbering).

In another aspect, the invention features an anti-FcRH5 antibody thatbinds to FcRH5 and CD3, wherein the anti-FcRH5 antibody comprises ananti-FcRH5 arm comprising a first binding domain comprising thefollowing six HVRs: (a) an HVR-H1 comprising the amino acid sequence ofSEQ ID NO: 1, (b) an HVR-H2 comprising the amino acid sequence of SEQ IDNO: 8, (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 9,(d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12, (e)an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (f)an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23, and ananti-CD3 arm comprising a second binding domain comprising the followingsix HVRs: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:115, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 116,(c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121, (d)an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 118, (e) anHVR-L2 comprising the amino acid sequence of SEQ ID NO: 119, and (f) anHVR-L3 comprising the amino acid sequence of SEQ ID NO: 123, and whereinthe anti-FcRH5 arm comprises a light chain comprising Q38E and V133Ksubstitution mutations and a heavy chain comprising Q39K, S183E, andN297G substitution mutations, and wherein the anti-CD3 arm comprises alight chain comprising Q38K, F116A, L135V, S174A, S176F, and T178Vsubstitution mutations and a heavy chain comprising Q39E, A141I, F170S,S181M, S183A, V185A, and N297G substitution mutations (EU numbering).

In another aspect, the invention features an anti-FcRH5 antibody thatbinds to FcRH5 and CD3, wherein the anti-FcRH5 antibody comprises: (a)an anti-FcRH5 arm comprising a first binding domain comprising a VHdomain comprising an amino acid sequence of SEQ ID NO: 104 and a VLdomain comprising an amino acid sequence of SEQ ID NO: 105, wherein theanti-FcRH5 arm comprises a light chain comprising Q38E and V133Ksubstitution mutations and a heavy chain comprising Q39K, S183E, andN297G substitution mutations, and (b) an anti-CD3 arm comprising asecond binding domain comprising a VH domain comprising an amino acidsequence of SEQ ID NO: 133 and a VL domain comprising an amino acidsequence of SEQ ID NO: 134, wherein the anti-CD3 arm comprises a lightchain comprising Q38K, F116A, L135V, S174A, S176F, and T178Vsubstitution mutations and a heavy chain comprising Q39E, A141I, F170S,S181M, S183A, V185A, and N297G substitution mutations (EU numbering).

In another aspect, the invention features an anti-FcRH5 antibody thatbinds to FcRH5 and CD3, wherein the anti-FcRH5 antibody comprises ananti-FcRH5 arm comprising a first binding domain comprising thefollowing six HVRs: (a) an HVR-H1 comprising the amino acid sequence ofSEQ ID NO: 1, (b) an HVR-H2 comprising the amino acid sequence of SEQ IDNO: 8, (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 9,(d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12, (e)an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (f)an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23, and ananti-CD3 arm comprising a second binding domain comprising the followingsix HVRs: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:115, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 116,(c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121, (d)an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 118, (e) anHVR-L2 comprising the amino acid sequence of SEQ ID NO: 119, and (f) anHVR-L3 comprising the amino acid sequence of SEQ ID NO: 123, and whereinthe anti-FcRH5 arm comprises a light chain comprising Q38K and V133Esubstitution mutations and a heavy chain comprising Q39E, S183K, andN297G substitution mutations, and wherein the anti-CD3 arm comprises alight chain comprising Q38E, F116A, L135V, S174A, S176F, and T178Vsubstitution mutations and a heavy chain comprising Q39K, A141I, F170S,S181M, S183A, V185A, and N297G substitution mutations (EU numbering).

In another aspect, the invention features an anti-FcRH5 antibody thatbinds to FcRH5 and CD3, wherein the anti-FcRH5 antibody comprises: (a)an anti-FcRH5 arm comprising a first binding domain comprising a VHdomain comprising an amino acid sequence of SEQ ID NO: 104 and a VLdomain comprising an amino acid sequence of SEQ ID NO: 105, wherein theanti-FcRH5 arm comprises a light chain comprising Q38K and V133Esubstitution mutations and a heavy chain comprising Q39E, S183K, andN297G substitution mutations, and (b) an anti-CD3 arm comprising asecond binding domain comprising a VH domain comprising an amino acidsequence of SEQ ID NO: 133 and a VL domain comprising an amino acidsequence of SEQ ID NO: 134, wherein the anti-CD3 arm comprises a lightchain comprising Q38E, F116A, L135V, S174A, S176F, and T178Vsubstitution mutations and a heavy chain comprising Q39K, A141I, F170S,S181M, S183A, V185A, and N297G substitution mutations (EU numbering).

In some embodiments of any one of the aspects of the invention, theanti-FcRH5 antibody has a clearance following intravenous injection ofbetween about 10 ml/kg/day to about 35 ml/kg/day. In some embodiments,the anti-FcRH5 antibody has a clearance following intravenous injectionof about 10 ml/kg/day to about 20 ml/kg/day in a mouse. In someembodiments, the anti-FcRH5 antibody has a clearance followingintravenous injection of about 12 ml/kg/day to about 16 ml/kg/day in amouse. In some embodiments, the anti-FcRH5 antibody has a clearancefollowing intravenous injection of about 20 ml/kg/day to about 40ml/kg/day in a cyno. In some embodiments, the anti-FcRH5 antibody has aclearance following intravenous injection of about 25 ml/kg/day to about35 ml/kg/day in a cyno. In some embodiments, the anti-FcRH5 antibody hasa clearance following intravenous injection of about 30 ml/kg/day toabout 35 ml/kg/day in a cyno.

In another aspect, the invention features an isolated nucleic acidencoding an anti-FcRH5 antibody of any one of the preceding aspects, ora portion thereof comprising a binding domain thereof that binds toFcRH5.

In another aspect, the invention features a vector comprising anisolated nucleic acid of the previous aspect.

In another aspect, the invention features a host cell comprising avector of the previous aspect. In some embodiments, the host cell is amammalian cell. In some embodiments, the mammalian cell is a Chinesehamster ovary (CHO) cell. In some embodiments, the host cell is aprokaryotic cell. In some embodiments, the prokaryotic cell is an E.coli cell.

In another aspect, the invention features a method of producing ananti-FcRH5 antibody of any one of the preceding aspects of theinvention, the method comprises culturing the host cell of the previousaspect in a culture medium. In some embodiments, the method furthercomprises recovering the anti-FcRH5 antibody from the host cell or theculture medium. In some embodiments, the method further comprisingculturing a second host cell comprising a second nucleic acid encodingan anti-CD3 antibody that comprises a binding domain that binds CD3. Insome embodiments, the host cells are co-cultured. In some embodiments,the method further comprises recovering the bispecific anti-FcRH5antibody from the host cells or the culture medium.

In another aspect, the invention features an immunoconjugate comprisingan anti-FcRH5 antibody of any one of the previous aspects and acytotoxic agent.

In another aspect, the invention features a composition comprising ananti-FcRH5 antibody of any one of the aspects of the invention. In someembodiments, the composition further comprising a pharmaceuticallyacceptable excipient or diluent. In some embodiments, thepharmaceutically acceptable excipient is a buffer, carrier, stabilizer,or preservative. In some embodiments, the composition is apharmaceutical composition. In some embodiments, the composition furthercomprises a PD-1 axis binding antagonist or an additional therapeuticagent.

In another aspect, the invention features an anti-FcRH5 antibody of anyone of the preceding aspects of the invention for use as a medicament.

In another aspect, the invention features an anti-FcRH5 antibody of anyone of the preceding aspects of the invention for use in treating ordelaying progression of an FcRH5-positive cancer in a subject in needthereof.

In another aspect, the invention features an anti-FcRH5 antibody of anyone of the preceding aspects of the invention for use in enhancingimmune function in a subject having an FcRH5-positive cancer. In someembodiments, the FcRH5-positive cancer is a B cell cancer. In anotheraspect, the B cell cancer is selected from the group consisting ofmultiple myeloma (MM), chronic lymphoid leukemia (CLL), mantle celllymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), and follicularlymphoma (FL). In another embodiment, the B cell cancer is MM.

In another aspect, the invention features the use of an anti-FcRH5antibody or a composition of any one of the previous aspects in themanufacture of a medicament for treating or delaying progression of anFcRH5-positive cancer in a subject. In another aspect, the inventionfeatures the use of an anti-FcRH5 antibody or a composition of any oneof the previous aspects in the manufacture of a medicament for enhancingimmune function in a subject having an FcRH5-positive cancer. In someembodiments, the FcRH5-positive cancer is a B cell cancer. In someembodiments, the B cell cancer is selected from the group consisting ofMM, CLL, MCL, DLBCL, and FL. In some embodiments, the B cell cancer isMM.

In another aspect, the invention features a method of treating ordelaying the progression of an FcRH5-positive cancer in a subject inneed thereof, the method comprising administering to the subject ananti-FcRH5 antibody of any one of the preceding aspects of theinvention. In another aspect, the invention features a method ofenhancing immune function in a subject having an FcRH5-positive cancer,the method comprising administering to the subject an effective amountof an anti-FcRH5 antibody of any one of the preceding aspects of theinvention. In some embodiments, the FcRH5-positive cancer is a B cellcancer. In some embodiments, the B cell cancer is selected from thegroup consisting of MM, CLL, MCL, DLBCL, and FL. In some embodiments,the B cell cancer is MM. In some embodiments, the anti-FcRH5 antibodybinds to (a) an FcRH5 molecule located on a target cell and (b) a CD3molecule located on an immune effector cell. In some embodiments, theanti-FcRH5 antibody activates the immune effector cell following bindingto the FcRH5 molecule and the CD3 molecule. In some embodiments, theactivated immune effector cell is capable of exerting a cytotoxic effectand/or an apoptotic effect on the target cell. In some embodiments, thetarget cell is a plasma cell. In some embodiments, the plasma cell is ashort-lived plasma cell. In some embodiments, the plasma cell is along-lived plasma cell. In some embodiments, the plasma cell is amyeloma cell. In some embodiments, the method comprises administering tothe subject the anti-FcRH5 antibody at a dosage of about 0.01 mg/kg/wkto about 50 mg/kg/wk. In some embodiments, the method comprisesadministering to the subject the anti-FcRH5 antibody at a dosage ofabout 0.1 mg/kg/wk to about 10 mg/kg/wk. In some embodiments, the methodcomprises administering to the subject the anti-FcRH5 antibody at adosage of about 1 mg/kg/wk.

In other embodiments, the method further comprising administering to thesubject a PD-1 axis binding antagonist and/or an additional therapeuticagent. In some embodiments, the PD-1 axis binding antagonist oradditional therapeutic agent is administered prior to or subsequent tothe administration of the anti-FcRH5 antibody. In some embodiments, thePD-1 axis binding antagonist or additional therapeutic agent isadministered concurrently with the anti-FcRH5 antibody. In someembodiments, the PD-1 axis binding antagonist is selected from the groupconsisting of a PD-L1 binding antagonist, a PD-1 binding antagonist, anda PD-L2 binding antagonist. In some embodiments, the PD-1 axis bindingantagonist is a PD-L1 binding antagonist. In some embodiments, the PD-L1binding antagonist is selected from the group consisting of MPDL3280A(atezolizumab), YW243.55.S70, MDX-1105, MED14736 (durvalumab), andMSB0010718C (avelumab). In some embodiments, the PD-L1 bindingantagonist is MPDL3280A (atezolizumab). In some embodiments, the PD-1axis binding antagonist is a PD-1 binding antagonist. In someembodiments, the PD-1 binding antagonist is selected from the groupconsisting of MDX 1106 (nivolumab), MK-3475 (pembrolizumab), CT-011(pidilizumab), MEDI-0680 (AMP-514), PDR001, REGN2810, and BGB-108. Insome embodiments, the PD-1 axis binding antagonist is a PD-L2 bindingantagonist. In some embodiments, the PD-L2 binding antagonist is anantibody or an immunoadhesin. In some embodiments, the subject asteroid, an immunomodulator (IMiD), a proteosome inhibitor (PI), or acombination thereof. In some embodiments, the steroid is aglucocorticoid. In some embodiments, the glucocorticoid isdexamethasone. In some embodiments, the IMiD is lenalidomide. In someembodiments, the PI is bortezomib.

In other embodiments, the method comprises administering the anti-FcRH5antibody, PD-1 axis binding antagonist, steroid, IMiD, PI, orcombination thereof, of any one of the preceding aspects, intravenously,subcutaneously, intramuscularly, topically, orally, transdermally,intraperitoneally, intraorbitally, by implantation, by inhalation,intrathecally, intraventricularly, or intranasally. In some embodiments,the method comprises administering the anti-FcRH5 antibody, PD-1 axisbinding antagonist, steroid, IMiD, PI, or combination thereof,intravenously. In some embodiments, the method comprises administeringthe anti-FcRH5 antibody, PD-1 axis binding antagonist, steroid, IMiD,PI, or combination thereof, subcutaneously. In some embodiments of anyone of the preceding aspects, the subject is a human.

In another aspect, the invention features a method for detecting FcRH5in a biological sample from a subject, wherein the method comprises: (a)contacting the biological sample with an anti-FcRH5 antibody of any oneof the aspects of the invention under conditions permissive for bindingof the anti-FcRH5 antibody to a naturally occurring FcRH5 in thebiological sample, and (b) detecting whether a complex is formed betweenthe anti-FcRH5 antibody and a naturally occurring FcRH5 in thebiological sample. In some embodiments, the biological sample is a bloodsample. In some embodiments of this aspect, the subject is a human.

In another aspect, the invention features a kit comprising an anti-FcRH5antibody of any one of the preceding aspects of the invention and apackage insert comprising instructions for using the anti-FcRH5 antibodyfor treating or delaying progression of an FcRH5-positive cancer in asubject. In another aspect, the invention features a kit comprising ananti-FcRH5 antibody of any one of the preceding aspects of the inventionand a package insert comprising instructions for using the anti-FcRH5antibody for enhancing immune function in a subject having anFcRH5-positive cancer. In some embodiments of these aspects, the subjectis a human.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic diagram showing an exemplary Rational Designconfiguration (Configuration 1) of an FcRH5 TDB having VL, VH, CL, andCH1 domains including one or more charged regions. For Configuration 1,the VH₁, CL₁, VL₂, and CH1₂ domains contain basic charged regions, andthe VL₁, CH1₁, VH₂, and CL₂ domains contain acidic charged regions.

FIG. 1B is a schematic diagram showing an exemplary Rational Designconfiguration (Configuration 2) of an FcRH5 TDB having VL, VH, CL, andCH1 domains including one or more charged regions. For Configuration 2,the VH₁, CL₁, VL₂, and CH1₂ domains contain acidic charged regions, andthe VL₁, CH1₁, VH₂, and CL₂ domains contain basic charged regions.

FIG. 1C is a schematic diagram showing an exemplary Rosetta Designconfiguration (Configuration 1) of an FcRH5 TDB having VL, VH, CL, andCH1 domains including one or more charged regions. For Configuration 1,the VH₁, CL₁, and VL₂ domains contain basic charged regions, and theVL₁, CH1₁, and VH₂ domains contain acidic charged regions. Additionally,the CH1₂ domain contains a cavity, and the CL₂ domain contains aprotuberance. The cavity and protuberance are depicted as a black box atthe CH1₂/CL₂ interface.

FIG. 1D is a schematic diagram showing an exemplary Rosetta Designconfiguration (Configuration 2) of an FcRH5 TDB having VL, VH, CL, andCH1 domains including one or more charged regions. For Configuration 2,the VH₁, CL₁, and VL₂ domains contain acidic charged regions, and theVL₁, CH1₁, and VH₂ domains contain basic charged regions. Additionally,the CH1₂ domain contains a cavity, and the CL₂ domain contains aprotuberance. The cavity and protuberance are depicted as a black box atthe CH1₂/CL₂ interface.

FIG. 1E is a schematic diagram showing an exemplary alternative RosettaDesign configuration (Alternative Configuration 1) of an FcRH5 TDBhaving VL, VH, CL, and CH1 domains including one or more chargedregions. For Alternative Configuration 1, the VL₁, VH₂, and CL₂ domainscontain acidic charged regions, and the VH₁, CH1₂, and VL₂ domainscontain basic charged regions. Additionally, the CH1₁ domain contains acavity, and the CL₁ domain contains a protuberance. The cavity andprotuberance are depicted as a black box at the CH1₁/CL₁ interface.

FIG. 1F is a schematic diagram showing an exemplary alternative RosettaDesign configuration (Alternative Configuration 2) of an FcRH5 TDBhaving VL, VH, CL, and CH1 domains including one or more chargedregions. For Alternative Configuration 2, the VL₁, VH₂, and CL₂ domainscontain basic charged regions, and the VH₁, CH1₂, and VL₂ domainscontain acidic charged regions. Additionally, the CH1₁ domain contains acavity, and the CL₁ domain contains a protuberance. The cavity andprotuberance are depicted as a black box at the CH1₁/CL₁ interface.

FIG. 2 is an alignment of the light chain variable (VL) domain sequencesof select anti-FcRH5 antibodies. Changes from 1G7 (see U.S. Pub. No2015-0098900, which is incorporated by reference herein in its entirety)are shown in dark boxes. Hypervariable regions (HVRs) are indicated bylines above and/or below the alignments. These VL domain sequences arealso disclosed as SEQ ID NOs: 83, 85, 87, 89, 91, 93, 95, 97, 99, and101.

FIG. 3 is an alignment of the heavy chain variable (VH) domain sequencesof select anti-FcRH5 antibodies. Changes from clone 1G7 (see U.S. Pub.No. 2015-0098900) are shown in dark boxes. Hypervariable regions (HVRs)are indicated by lines above and/or below the alignments. These VHdomain sequences are also disclosed as SEQ ID NOs: 82, 84, 86, 88, 90,92, 94, 96, 98, and 100.

FIG. 4 is a table showing the impact on binding affinity of amino acidsubstitutions at position 52 of the indicated anti-FcRH5 antibodies.

FIG. 5A is an alignment of the heavy chain variable (VH) domainsequences of hu1G7.v85 and hu1G7.v93. Changes from the human germlinesequence hlGHV4-59*01 are shown in shaded boxes. Hypervariable regions(HVRs) are indicated by labels above the alignments. These VH domainsequences are also disclosed as SEQ ID NOs: 104 (hu1G7.v85) and 106(hu1G7.v93).

FIG. 5B is an alignment of the light chain variable (VL) domainsequences of hu1G7.v85 and hu1G7.v93. Changes from the human germlinesequence hlGKV1-16*01 are shown in shaded boxes. Hypervariable regions(HVRs) are indicated by labels above the alignments. These VL domainsequences are also disclosed as SEQ ID NOs: 105 (hu1G7.v85) and 107(hu1G7.v93).

FIG. 6A is an alignment of the heavy chain variable (VH) domainsequences of hu1G7.v85. 1G7, and consensus H4. Changes from thehumanized, affinity matured, and polished clone 1G7 (see U.S. Pub. No.2015-0098900, which is incorporated by reference herein in its entirety)are shown in shaded boxes. Hypervariable regions (HVRs) are indicated bylabels above the alignments. The VH domain sequence of hu1G7.v85 isdisclosed as SEQ ID NO: 104.

FIG. 6B is an alignment of the light chain variable (VL) domainsequences of hu1G7.v85, 1G7, and consensus KI. Changes from thehumanized, affinity matured, and polished clone 1G7 are shown in shadedboxes. The VL domain sequence of hu1G7.v85 is disclosed as SEQ ID NO:105.

FIG. 7A shows the heavy chain variable (VH) domain sequence ofanti-FcRH5 antibody hu1G7.v93 (SEQ ID NO: 106).

FIG. 7B shows the light chain variable (VL) domain sequence ofanti-FcRH5 antibody hu1G7.v93 (SEQ ID NO: 107).

FIG. 8 is a graph showing the antibody titer of 1G7.v85 and 1G7.v87antibodies.

FIG. 9A is an overlay of histograms comparing the binding ofFcRH5/38E4.v1 TDBs having different anti-FcRH5 arms (i.e., m1G7 (“1G7TDB”), 1G7.v85 (“1G7.v85 TDB”), and 1G7.v1.4 (“1G7.v1.4 TDB”)) toFcRH3-overexpressing cells.

FIG. 9B is a graph showing that the 1G7.v85 TDB does not deplete naturalkiller (NK) cells at concentrations of ≤20 μg/mL; 1G7.v85 TDB has amedian EC50 of 25 ng/mL on plasma cells (PCs).

FIG. 10A is a series of graphs comparing the ability of the FcRH5 TDB1G7.v1.4/38E4.v1 (“1G7.v1.4 TDB”) to bind FcRH3, human FcRH5, and cynoFcRH5.

FIG. 10B is a series of graphs comparing the ability of the 1G7.v85 TDBto bind FcRH3, human FcRH5, and cyno FcRH5.

FIG. 10C is a series of graphs comparing the ability of the FcRH5 TDB1G7/38E4.v1 (“1G7 TDB”) to bind FcRH3, human FcRH5, and cyno FcRH5.

FIG. 10D is a series of graphs comparing the ability of a controlantibody to bind FcRH3, human FcRH5, and cyno FcRH5.

FIG. 11A shows the sequence of the heavy chain variable (VH) domainsequence of humanized, rabbit-derived anti-FcRH5 antibody hu7D8.L1H2.The VH domain sequence of hu7D8.L1H2 is disclosed as SEQ ID NO: 108.

FIG. 11B shows the sequence of the light chain variable (VL) domainsequence of humanized, rabbit-derived anti-FcRH5 antibody hu7D8.L1H2.The VL domain sequence of hu7D8.L1H2 is disclosed as SEQ ID NO: 109.

FIG. 12A shows the sequence of the heavy chain variable (VH) domainsequences of mouse-derived anti-FcRH5 antibody 17B1. The VH domainsequences of 17B1 is disclosed as SEQ ID NO: 110.

FIG. 12B shows the sequence of the light chain variable (VL) domainsequence of mouse-derived anti-FcRH5 antibody 17B1. The VL domainsequences of 17B1 is disclosed as SEQ ID NO: 111.

FIG. 13A shows the sequence of the heavy chain variable (VH) domainsequences of mouse-derived anti-FcRH5 antibody 15G8. The VH domainsequence of 15G8 is disclosed as SEQ ID NO: 112.

FIG. 13B shows the sequence of the light chain variable (VL) domainsequence of mouse-derived anti-FcRH5 antibody 15G8. The VL domainsequence of 15G8 is disclosed as SEQ ID NO: 113.

FIG. 14 is a series of graphs showing the binding affinity of humanizedvariant of rabbit-derived anti-FcRH5 antibodies 7D8.Rb and h7D8.L1H2 tohuman FcRH5 and cyno FcRH5.

FIG. 15 is a series of graphs showing that the FcRH5 TDBs 1G7.v85 TDBand hu1G7.v93/38E4.v1 (“1G7.v93 TDB”) bind with comparable affinity toboth human FcRH5 and cyno FcRH5.

FIGS. 16A-16B are graphs showing the kinetic analysis of 1G7.v85 TDBbinding to human FcRH5 and cyno FcRH5 with dissociation constants(K_(D)) of 2.35 nM and 6.76 nM, respectively, as measured by BIACORE® inan hlgG capture format using a 1:1 binding model of monovalent affinity.

FIGS. 17A-17B are graphs demonstrating increased FcRH5-induced celltoxicity of MOLP-2 cells (i.e., human multiple myeloma cells thatendogenously express FcRH5) using humanized and affinity maturedvariants of 1G7 formatted into T cell-dependent bispecific (TDB)antibodies having the CD3-binding arm of 38E4.v1 (see PCT Pub. No. WO2015-095392 A1, which is incorporated by reference herein in itsentirety). In FIG. 17A, the 1G7 TDB, hu1G7.v1.1/38E4.v1 (“1G7.v1.1TDB”), hu1G7.v1.2/38E4.v1 (“1G7.v1.2 TDB”), hu1G7.v1.3/38E4.v1(“1G7.v1.3 TDB”), and 1G7.v1.4 TDB were evaluated. In FIG. 17B, the1G7.v1.4 TDB, hu1G7.v1.5/38E4.v1 (“1G7.v1.5” TDB), hu1G7.v1.13/38E4.v1(“1G7.v1.13 TDB”), hu1G7.v1.7/38E4.v1 (“1G7.v1.7 TDB”), andhu1G7.v1.13.1/38E4.v1 (“1G7.v1.13.1”) were evaluated. The 1G7.v.1.4 TDBimproved target cell killing (EC50) 5- to 13-fold over murine 1G7 TDB(n=10).

FIGS. 18A-18D are graphs comparing the ability of the 1G7.v1.4 TDB andthe 1G7.v85 TDB to activate T cells (FIG. 18A), kill target MOLP-2 cells(FIG. 18B), kill target cyno plasma cells (FIG. 18C), and kill targetcyno B cells (FIG. 18D).

FIG. 19A is a series of histograms comparing the ability of the 1G7.v85TDB and the hu1G7.v87/38E4.v1 TDB (“1G7.v87 TDB”) to bind to andcross-react with mouse SVT2 cells expressing human FcRH5 (Panel 1), cynoFcRH5 (Panel 2), and human FcRH3 (Panel 3).

FIGS. 19B-19D are graphs comparing the ability of the 1G7.v85 TDB andthe 1G7.v87 TDB to kill target MOLP-2 cells (FIG. 19B), human B cells(FIG. 19C), and cyno B cells (FIG. 19D).

FIGS. 20A-20D are graphs showing a reduced ability of the 1G7.v85 TDB tobind FcRH5 after undergoing either a2,2′-azobis(2-amidopropane)dihydrochloride (AAPH) stress test (FIG. 20B)or a light stress test (FIG. 20D), as compared to unstressed respectivecontrols (FIGS. 20A and 20C).

FIG. 21A is graph showing size distribution analysis of the 1G7.v85 TDB.The 1G7.v85 TDB lost 0.1% of the monomer peak after two weeks of stressin a his-acetate solution at pH 5.5.

FIG. 21B is a graph showing the charge heterogeneity of the 1G7.v85 TDB.The 1G7.v85 TDB lost 7.7% of the monomer peak after two weeks of stressin a his-acetate solution at pH 5.5 FIG. 22A is a graph showing that the1G7.v85 TDB has no observable change in the reduced mass profile of thelight chain mass after two weeks of stress in a his-acetate solution atpH 5.5.

FIG. 22B is a graph showing that the 1G7.v85 TDB has no observablechange in the reduced mass profile of the heavy chain mass after twoweeks of stress in a his-acetate solution at pH 5.5.

FIG. 23A is a phospho-SLP76 Western blot of a healthy donor peripheralCD8 cells stimulated with 1 μg/ml of the 1G7/UCHT1.v9 TDB, 10A8/UCHT1.v9TDB (“10A8 TDB”), and anti-gD/UCHT1.v9 TDB (“anti-gD TDB”), and cellsexpressing human FcRH5 with N-terminal gD expression tag. Blotting fortotal SLP76, indicative of TCR signaling, was used to confirm equalsample loading.

FIG. 23B is a graph showing FcRH5 target cell killing with either1G7/UCHT1.v9 TDB or anti-gD TDB and CD8+ T cells.

FIG. 23C is a schematic diagram of the truncated FcRH5 construct withthe gD epitope now membrane-proximal.

FIG. 23D is a graph showing target cell killing using the truncatedFcRH5 construct with 1G7 TDB or anti-gD TDB. The activity of theproximal 1G7/UCHT1.v9 TDB increased by 25-fold (EC50=20 pM), and theanti-gD TDB was able to effectively mediate killing of cells (EC50=0.19nM) when the interference caused by the ECD was removed. The truncatedconstruct was expressed in 293 cells.

FIG. 23E is a graph showing that target cell killing for five alternateFcRH5 TDBs that recognize the membrane-proximal epitope (dashed lines)are equivalent to killing mediated by the 1G7/UCHT1.v9 TDB andsignificantly better than the 10A8 TDB.

FIG. 24A is a histogram overlay of flow cytometry analysis ofSVT2-parental, gD-FcRH5 full-length, and gD-FcRH5-domain 9 cells.

FIG. 24B is a graph showing percent target cell killing by 1G7/UCHT1.v9TDB, 2H7/UCHT1.v9 TDB (“2H7 TDB”), 3G7/UCHT1.v9 TDB (“3G7 TDB”), 10A8TDB, and anti-gD TDB.

FIG. 25A is an overlay histogram of six cell lines (SVT2-vector,SVT2-FcRH1, SVT2-FCRH2, SVT2-FcRH3, SVT2-FcRH4, and SVT2-FcRH5), showingthat the 1G7.v85 TDB binds to FcRH5, but not to other family members.

FIG. 25B is a histogram overlay of three cell lines (293 parental,293-FcRH5 full-length, and 293-FcRH5-D9-deletion), showing that the1G7.v85 TDB binds to the membrane proximal domain of FcRH5.

FIG. 25C is an overlay histogram of three cell lines (SVT2-vector,SVT2-huFcRH5, and SVT2-cyno FcRH5), showing that the 1G7.v85 TDB bindsto cyno FcRH5 and human FcRH5.

FIGS. 26A-26D are overlay histograms of three cell lines (SVT2-vector,SVT2-huFcRH5, and SVT2-cyno FcRH5), showing binding of the 1G7/38E4.v1TDB (“1G7 TDB”) to multiple myeloma (MM) cell line and primary cells.Overlay histograms are shown for isotype-PE and 1G7 TDB for MOLP-2 cells(FIG. 26A), human CD20+ B cells (FIG. 26B), human CD38+CD138+ plasmacells (FIG. 26C), and CD38+CD138+MM tumor cells from MM bone marrowaspirate (FIG. 26D).

FIG. 27A is a graph showing dose-dependent activation of CD8+ cells uponstimulation with target cells (MOLP-2) and 1G7 TDB, detected by flowcytometry analysis.

FIG. 27B is a graph showing target cell-dependent killing by 1G7 TDB.The insert shows a flow cytometry overlay of the parental Fox-NY cellline and clones transfected to express a low or a high level of humanFcRH5. Error bars are standard deviation of triplicates.

FIGS. 27C-27E are graphs showing that 1G7 TDB induced CD8 proliferationresponse (5 days), as detected by measuring CSFE fluorescence intensitydilution by flow cytometry. CFSE-labeled human CD8+ cells only (FIG.27C), co-culture with MOLP-2 (FIG. 27D), or co-culture with MOLP-2 and1000 ng/ml of the 1G7.v85 TDB (FIG. 27E).

FIG. 27F is a graph showing target-dependent cell killing with FcRH5TDBs containing different anti-CD3 arms (e.g., UCHT1.v9, 38E4.v1, and40G5c) (see PCT Pub. No. WO 2015/095392 A1, which is incorporated byreference herein in its entirety). The anti-CD3 arms 38E4.v1 (monovalentK_(D) 0.5 nM, BIACORE®), UCHT1.v9 (monovalent K_(D)=2.5 nM, BIACORE® andScatchard), and 40G5c (monovalent K_(D)=51 nM, BIACORE®) were eachpaired with the anti-FcRH5 arm of ml G7 (K_(D)=11 nm, BIACORE®) andtested for binding to purified human CD8+ cells.

FIG. 27G is a graph showing target-dependent cell killing with the 1G7TDB and 1G7/38E4.v1 bis-Fab (“1G7 bis-Fab”).

FIG. 28A is a graph showing FcRH5 expression in primary multiple myelomatumor cells, healthy donor peripheral B cells, and bone marrow plasmacells. FcRH5 expression was analyzed by flow cytometry and normalized toexpression in MOLP-2 internal and assay control. The relative level ofFcRH5 was calculated as follows: (Geometric mean of FcRH5 of“X”/Geometric mean of isotype control of “X”)/(Geometric mean of FcRH5of MOLP-2/Geometric mean of isotype control of MOLP-2).

FIG. 28B is a graph showing cytotoxic activity of the 1G7.v85 TDB onhuman plasma cells. Human bone marrow mononuclear cells (BMMCs) werecultured with 1G7.v85 TDB and the cell number of live CD38+CD138+ wasanalyzed by flow cytometry.

FIG. 28C is a graph showing the cytotoxic activity of the 1G7.v85 TDB ondifferent patient-derived primary myeloma cells. Human myeloma BMMCswere co-cultured with CD8+ T cells isolated from healthy donor and1G7.v85 TBD.

FIG. 28D is a graph showing that very low FcRH5 expression in targetcells is sufficient for potent killing activity. FcRH5 copy number percell was determined by Scatchard assay.

FIG. 28E (Top) is a graph showing qRT-PCR analysis of FcRH5 mRNA levelsin bone marrow biopsies from high risk myeloma patients with 1q21 gain.mRNA expression level was calculated by the delta Ct (dCt) method.Statistical analysis was done using a Mann-Whitney U test. (Bottom) Areimages of FISH analysis on primary multiple myeloma biopsies showingnormal diploid of 1q21 (Left) and a mixture of approximately three tosix copies of 1q21 (Right). A tumor sample was identified as 1q21 gainwhen >20% of the tumor cells scored had three or more copies of the1q21.3 locus.

FIG. 29A is a histogram overlay of isotype-PE and anti-FcRH5 clone1G7-PE, depicting the expression of FcRH5 on cyno CD20+ B cells.

FIG. 29B is a histogram overlay of isotype-PE and anti-FcRH5 clone1G7-PE, depicting the expression of FcRH5 on cyno on CD45-CD20-CD38+PC+plasma cells.

FIGS. 29C-29D are graphs showing comparable dose-dependent cytotoxicactivity between cyno CD8+ T cells and human CD8+ T cells in an in vitrokilling assay using SVT2-cyno-FcRH5 (FIG. 29C) and MOLP-2 (FIG. 29D)with human CD8+ T cells or cyno CD8+ T cells.

FIG. 29E is a graph showing in vitro killing activity of 1G7.v85 TDB oncyno CD20+ B cells (n=14).

FIG. 29F is a graph showing in vitro killing activity of 1G7.v85 TDB onCD45-CD20-CD38+PC+ plasma cells from cyno bone marrow from eightdifferent donors (n=8).

FIG. 30A is a graph showing that splenic human T cells isolated fromspleens of humanized NOD/SCID gamma mice (NSG) have comparable activityto peripheral human T cells from heathy donors.

FIG. 30B is a graph showing that 1G7 TDB treatment induces regression ofsubcutaneous MOLP-2 xenograft tumors in humanized NSG mice. The micewere treated with a single intravenous dose of vehicle or 1G7.v85 TDB at0.5 mg/kg. Mean tumor volume (black bold line), the individual tumorvolumes (thin lines), and mean of control group (dashed line) areindicated.

FIG. 31A is a graph showing the serum concentration of FcRH5 TDB plottedover the duration of the study after single-dose administration of1G7.v85 TDB at 1 mg/kg, 2 mg/kg, or 4 mg/kg to three animals/group. Atable showing pharmacodynamics parameters is shown below.

FIG. 31B is a graph showing the serum concentration of FcRH5 TDBs(1G7.v85 TDB and 1G7.v87/38E4.v1 TDB (“1G7.v87 TDB”)) plotted over theduration of study after single dose administration of anti-gD TDB at 3mg/kg or FcRH5 TDBs at 0.3 mg/kg or 3 mg/kg to three animals/group. Atable showing pharmacodynamics parameters is shown below.

FIGS. 31C-31D are graphs showing 1G7.v85 TDB-induced transient T cellactivation in cyno peripheral blood after single-dose intravenousadministration of vehicle or 1G7.v85 TDB (1 mg/kg, 2 mg/kg, or 4 mg/kg)to three animals/group.

FIGS. 31E-31H are graphs showing the absolute count of CD20+ B cells inperipheral blood (FIG. 31E), spleen (FIG. 31F), mandibular lymph node(FIG. 31G), and bone marrow (FIG. 31H) in cyno after single-doseintravenous administration of vehicle or 1G7.v85 TDB (1 mg/kg, 2 mg/kg,or 4 mg/kg) to three animals/group. FIGS. 31F-31H are plotted with groupmean and standard error of mean (SEM).

FIG. 31I is a graph showing that 1G7.v85 TDB depletes bone marrow plasmacells in cyno, with group measured SEM plotted.

FIG. 31J is a graph showing the change of cyno IgG level response totreatment, calculated using formula {(IgG level pre-dose)−(IgG level endof study)}/(IgG level pre-dose)×100. The difference between pre-dose andafter treatment is analyzed by an unpaired t-test. The data are plottedwith group mean and standard error of mean (SEM).

FIGS. 32A-32B are graphs showing the absolute count of CD4+ T cells inperipheral blood (FIG. 32A) and CD8+ T cells (FIG. 32B) in four groupsof animals after single-dose intravenous administration of vehicle,1G7.v85 TDB at 1 mg/kg, 1G7.v85 TDB at 2 mg/kg, or 1G7.v85 TDB at 4mg/kg.

FIG. 32C is a graph showing the decrease in the absolute count of CD20+B cells in mesenteric lymph nodes after 1G7.v85 TDB treatment. The plotis graphed as individual animals and mean group with SEM.

FIG. 32D is a graph showing FcRH5 occupancy after single-doseintravenous administration of 1G7.v85 TDB at 1 mg/kg, 2 mg/kg, or 4mg/kg.

FIGS. 33A-33F are graphs showing the change in concentration of thecytokines IL-6 (FIG. 33A), IL-2 (FIG. 33B), IFN-γ (FIG. 33C), IL-1Rα(FIG. 33D), IL-5 (FIG. 33E), and MCP-1 (FIG. 33F) in four groups ofanimals after single-dose intravenous administration of vehicle, 1G7.v85TDB at 1 mg/kg, 1G7.v85 TDB at 2 mg/kg, and 1G7.v85 TDB at 4 mg/kg.

FIG. 34 is a series of plots showing that 1G7.v85 TDB treatment inducesPD1 expression in human T cells. CD8+ T cells were stimulated for 48hours with the 1G7.v85 TDB and MOLP-2 target cells and then analyzed byflow cytometry.

FIG. 35A is a graph showing the percentage of PD1+ in CD8+ T cells incyno after a single-dose intravenous administration of vehicle, 1 mg/kg,2 mg/kg, and 4 mg/kg of 1G7.v85 TDB. 1G7.v85 TDB treatment results ininduction of PD1 in cyno T cells in vivo.

FIG. 35B is a graph showing the percentage of PD1+ in CD4+ T cells incyno after single dose intravenous administration of vehicle, 1 mg/kg, 2mg/kg, and 4 mg/kg of 1G7.v85 TDB.

FIGS. 36A-36D are plots showing PD-1 expression in CD4+ T cells fromblood (FIG. 36A), CD8+ T cells from spleen (FIG. 36B), CD8+ T cells fromlymph node (FIG. 36C), and CD8+ T cells from bone marrow (FIG. 36D), asanalyzed by FACS seven days after dosing with 1G7.v85 TDB or vehicle.

FIGS. 37A-37B are graphs showing the ability of the 1G7.v85 TDB toredirect activity of pre-stimulated CD8+ T cells to kill HEK-293T cellsexpressing FcRH5 and PD-1 (“293-FcRH5-PD-L1 cells”) in the presence orabsence of an anti-PD-L1 or anti-PD-1 antibody. The curve in FIG. 37A isgraphed with mean and standard of error (SD) of triplicates.

FIG. 38A is a graph showing target cell killing of SVT2-FcRH5 by 1G7.v85TDB in the presence and absence of dexamethasone (Dex).

FIG. 38B is a series of graphs showing cytokine (i.e., IL-2, IL-6,TNF-α, and IFN-γ) release after treatment with 1G7.v85 TDB in thepresence and absence of 1 pM Dex.

FIG. 39 is a schematic drawing showing the production of bis-Fabs fromhuman IgG1.

FIG. 40A is a graph showing the binding of FcRH5 by bis-Fabs A-D andF(ab′)₂ A as determined by an ELISA assay.

FIG. 40B is a graph showing the binding of CD3 by bis-Fabs A-D andF(ab′)₂ A as determined by an ELISA assay.

FIG. 41 is a graph showing the amount of target-mediated T cellactivation by bis-Fabs A-D and F(ab′)₂ A.

FIG. 42 is a table showing the corresponding HVR-L1, HVR-L2, and HVR-L3sequences of each antibody clone examined by surface plasmon resonancealong with K_(D) (nM) and fold improvement of the particular clones ascompared to hu1G7.v1 controls.

FIG. 43 is a table showing the corresponding HVR-H1, HVR-H2, and HVR-H3sequences of each antibody clone examined by surface plasmon resonancealong with K_(D) (nM) and fold improvement of the particular clones ascompared to hu1G7.v1 controls.

FIG. 44 is a table showing combinatorial analysis of mutationsidentified by antibody phage display.

FIG. 45 is a table showing enrichment scores of mutations in hu1G7.v1selected with human FcRH5.

FIG. 46 is a table showing enrichment scores of mutations in hu1G7.v1selected with cynomolgous FcRH5.

FIG. 47 is a table showing mutations with enrichment scores of at least0.5 in selections with human FcRH5 and enrichment scores of at least 0in selections with cynomolgous FcRH5. The mutations selected for furtheranalysis are highlighted in black or gray, and the L29T mutationidentified by Sanger sequencing is highlighted in gray.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The term “about” as used herein refers to the usual error range for therespective value readily known to the skilled person in this technicalfield. Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse.

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencechanges. In some embodiments, the number of amino acid changes are 10 orfewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 orfewer, 3 or fewer, or 2 or fewer. In some embodiments, the VL acceptorhuman framework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

“Affinity” refers to the strength of the sum total of noncovalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., antibody and antigen). The affinity of a molecule Xfor its partner Y can generally be represented by the dissociationconstant (K_(D)). Affinity can be measured by common methods known inthe art, including those described herein. Specific illustrative andexemplary embodiments for measuring binding affinity are described inthe following.

An “affinity matured” antibody refers to an antibody with one or morealterations in one or more hypervariable regions (HVRs), compared to aparent antibody which does not possess such alterations, suchalterations resulting in an improvement in the affinity of the antibodyfor antigen.

The term “anti-FcRH5 antibody” or “an antibody that binds to FcRH5”refers to an antibody that is capable of binding FcRH5 with sufficientaffinity such that the antibody is useful as a diagnostic and/ortherapeutic agent in targeting FcRH5. In one embodiment, the extent ofbinding of an anti-FcRH5 antibody to an unrelated, non-FcRH5 protein isless than about 10% of the binding of the antibody to FcRH5 as measured,e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibodythat binds to FcRH5 has a dissociation constant (K_(D)) of ≤1 μM, ≤100nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10⁻⁸ M orless, e.g., from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ M to 10⁻¹³ M). Incertain embodiments, an anti-FcRH5 antibody binds to an epitope of FcRH5that is conserved among FcRH5 from different species.

The terms “anti-CD3 antibody” and “an antibody that binds to CD3” referto an antibody that is capable of binding CD3 with sufficient affinitysuch that the antibody is useful as a diagnostic and/or therapeuticagent in targeting CD3. In one embodiment, the extent of binding of ananti-CD3 antibody to an unrelated, non-CD3 protein is less than about10% of the binding of the antibody to CD3 as measured, e.g., by aradioimmunoassay (RIA). In certain embodiments, an antibody that bindsto CD3 has a dissociation constant (K_(D)) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10⁻⁸ M or less, e.g., from10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ M to 10⁻¹³ M). In certainembodiments, an anti-CD3 antibody binds to an epitope of CD3 that isconserved among CD3 from different species.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments (e.g., bis-Fabs) so longas they exhibit the desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to bis-Fabs; Fv; Fab; Fab,Fab′-SH; F(ab′)₂; diabodies; linear antibodies; single-chain antibodymolecules (e.g., scFv); and multispecific antibodies formed fromantibody fragments.

By “binding domain” is meant a part of a compound or a molecule thatspecifically binds to a target epitope, antigen, ligand, or receptor.Binding domains include but are not limited to antibodies (e.g.,monoclonal, polyclonal, recombinant, humanized, and chimericantibodies), antibody fragments or portions thereof (e.g., bis-Fabfragments, Fab fragments, F(ab′)₂, scFv antibodies, SMIP, domainantibodies, diabodies, minibodies, scFv-Fc, affibodies, nanobodies, andVH and/or VL domains of antibodies), receptors, ligands, aptamers, andother molecules having an identified binding partner.

As used herein the term “charged region” refers to a location of apolypeptide (e.g., an antibody) that includes one or more (e.g., 1, 2,3, 4, 5, 6, 7, 8, 9, or 10) basic or acidic amino acids that are capableof forming a charge pair with a cognate charged region having one ormore (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) or basic or acidic aminoacids, when the charged region and its cognate charged region haveopposite overall relative charge.

As used herein the term “charge pair” refers to the bond that is formedbetween two charged regions of opposite overall charge.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN®);alkyl sulfonates such as busulfan, improsulfan and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; acetogenins(especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol(dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinicacid; a camptothecin (including the synthetic analogue topotecan(HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin,scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); podophyllotoxin; podophyllinic acid; teniposide;cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogues, KW-2189 andCB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,chlorophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gamma1I and calicheamicinomegall (see, e.g., Nicolaou et al. Angew. Chem Intl. Ed. Engl. 33:183-186, 1994); CDP323, an oral alpha-4 integrin inhibitor; dynemicin,including dynemicin A; an esperamicin; as well as neocarzinostatinchromophore and related chromoprotein enediyne antibiotic chromophores),aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, carabicin, caminomycin, carzinophilin, chromomycins,dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,doxorubicin (including ADRIAMYCIN®, morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HClliposome injection (DOXIL®), liposomal doxorubicin TLC D-99 (MYOCET®),peglylated liposomal doxorubicin (CAELYX®), and deoxydoxorubicin),epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such asmitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin,porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;anti-metabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur(UFTORAL®), capecitabine (XELODA®), an epothilone, and 5-fluorouracil(5-FU); combretastatin; folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS NaturalProducts, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium;tenuazonic acid; triaziquone; 2,2′,2′-trichlorotriethylamine;trichothecenes (especially T-2 toxin, verracurin A, roridin A andanguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); thiotepa; taxoid, e.g., paclitaxel (TAXOL®,Bristol-Myers Squibb Oncology, Princeton, N.J.), albumin-engineerednanoparticle formulation of paclitaxel (ABRAXANE™), and docetaxel(TAXOTERE®, Rhome-Poulene Rorer, Antony, France); chloranbucil;6-thioguanine; mercaptopurine; methotrexate; platinum agents such ascisplatin, oxaliplatin (e.g., ELOXATIN®), and carboplatin; vincas, whichprevent tubulin polymerization from forming microtubules, includingvinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®,FILDESIN®), and vinorelbine (NAVELBINE®); etoposide (VP-16); ifosfamide;mitoxantrone; leucovorin; novantrone; edatrexate; daunomycin;aminopterin; ibandronate; topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoids such as retinoic acid,including bexarotene (TARGRETIN®); bisphosphonates such as clodronate(for example, BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095,zoledronic acid/zoledronate (ZOMETA®), alendronate (FOSAMAX®),pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®);troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisenseoligonucleotides, particularly those that inhibit expression of genes insignaling pathways implicated in aberrant cell proliferation, such as,for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor(EGF-R) (e.g., erlotinib (Tarceva™)); and VEGF-A that reduce cellproliferation; vaccines such as THERATOPE® vaccine and gene therapyvaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, andVAXID® vaccine; topoisomerase 1 inhibitor (e.g., LURTOTECAN®); rmRH(e.g., ABARELIX®); BAY439006 (sorafenib; Bayer); SU-11248 (sunitinib,SUTENT®, Pfizer); perifosine, COX-2 inhibitor (e.g., celecoxib oretoricoxib), proteosome inhibitor (e.g., PS341); bortezomib (VELCADE®);CCI-779; tipifarnib (R11577); orafenib, ABT510; Bcl-2 inhibitor such asoblimersen sodium (GENASENSE®); pixantrone; EGFR inhibitors; tyrosinekinase inhibitors; serine-threonine kinase inhibitors such as rapamycin(sirolimus, RAPAMUNE®); farnesyltransferase inhibitors such aslonafarnib (SCH 6636, SARASAR™); and pharmaceutically acceptable salts,acids or derivatives of any of the above; as well as combinations of twoor more of the above such as CHOP, an abbreviation for a combinedtherapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone;and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin(ELOXATIN™) combined with 5-FU and leucovorin, and pharmaceuticallyacceptable salts, acids or derivatives of any of the above; as well ascombinations of two or more of the above.

Chemotherapeutic agents as defined herein include “anti-hormonal agents”or “endocrine therapeutics” which act to regulate, reduce, block, orinhibit the effects of hormones that can promote the growth of cancer.They may be hormones themselves, including, but not limited to:anti-estrogens and selective estrogen receptor modulators (SERMs),including, for example, tamoxifen (including NOLVADEX® tamoxifen),raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene,LY117018, onapristone, and FARESTON.cndot.toremifene; aromataseinhibitors that inhibit the enzyme aromatase, which regulates estrogenproduction in the adrenal glands, such as, for example, 4(5)-imidazoles,aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane,formestanie, fadrozole, RIVISOR® vorozole, FEMARA® letrozole, andARIMIDEX® anastrozole; and anti-androgens such as flutamide, nilutamide,bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides,particularly those which inhibit expression of genes in signalingpathways implicated in abherant cell proliferation, such as, forexample, PKC-alpha, Raf and H-Ras; ribozymes such as a VEGF expressioninhibitor (e.g., ANGIOZYME® ribozyme) and a HER2 expression inhibitor;vaccines such as gene therapy vaccines, for example, ALLOVECTIN®vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; PROLEUKIN® rlL-2;LURTOTECAN® topoisomerase 1 inhibitor; ABARELIX® rmRH; Vinorelbine andEsperamicins (see U.S. Pat. No. 4,675,187), and pharmaceuticallyacceptable salts, acids or derivatives of any of the above; as well ascombinations of two or more of the above.

The term “immunomodulatory drug” or “IMiD” refers to a class of drugsthat modifies the immune system response or the functioning of theimmune system, such as by the stimulation of antibody formation and/orthe inhibition of peripheral blood cell activity, and include, but arenot limited to, thalidomide (α-N-phthalimido-glutarimide) and itsanalogues, REVLIMID® (lenalidomide), ACTI-MID™ (pomalidomide), OTEZLA®(apremilast), and pharmaceutically acceptable salts or acids thereof.

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The term “FcRH5,” as used herein, refers to any native FcRH5 whichresults from the production of a FcRH5 protein in a cell. The termincludes FcRH5 from any vertebrate source, including mammals such asprimates (e.g., humans and cynomolgus monkeys) and rodents (e.g., miceand rats), unless otherwise indicated. The term also includes naturallyoccurring variants of FcRH5, e.g., splice variants or allelic variants.The amino acid sequence of an exemplary human FcRH5 protein sequence isshown in SEQ ID NO: 114. The amino acid sequence of an exemplarycynomolgus monkey FcRH5 protein is shown in SEQ ID NO: 215.

The term “cluster of differentiation 3” or “CD3,” as used herein, refersto any native CD3 from any vertebrate source, including mammals such asprimates (e.g., humans) and rodents (e.g., mice and rats), unlessotherwise indicated, including, for example, CD3ε, CD3γ, CD3α, and CD3βchains. The term encompasses “full-length,” unprocessed CD3 (e.g.,unprocessed or unmodified CD3ε or CD3γ), as well as any form of CD3 thatresults from processing in the cell. The term also encompasses naturallyoccurring variants of CD3, including, for example, splice variants orallelic variants. CD3 includes, for example, human CD3ε protein (NCBIRefSeq No. NP_000724), which is 207 amino acids in length, and humanCD3γ protein (NCBI RefSeq No. NP_000064), which is 182 amino acids inlength.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ, respectively.

It is understood that aspects and embodiments of the invention describedherein include “comprising,” “consisting,” and “consisting essentiallyof” aspects and embodiments.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents a cellular function and/or causes cell death ordestruction. Cytotoxic agents include, but are not limited to,radioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³,Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu); chemotherapeuticagents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids(vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycinC, chlorambucil, daunorubicin or other intercalating agents); growthinhibitory agents; enzymes and fragments thereof such as nucleolyticenzymes; antibiotics; toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof; and the variousantitumor or anticancer agents disclosed below.

A “disorder” is any condition that would benefit from treatmentincluding, but not limited to, chronic and acute disorders or diseasesincluding those pathological conditions which predispose the mammal tothe disorder in question.

The terms “cell proliferative disorder” and “proliferative disorder”refer to disorders that are associated with some degree of abnormal cellproliferation. In one embodiment, the cell proliferative disorder iscancer. In one embodiment, the cell proliferative disorder is a tumor.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth/proliferation. Examples of cancer include, butare not limited to, myeloma, carcinoma, lymphoma (e.g., Hodgkin's andnon-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. In someembodiments, the cancer is an FcRH5-positive cancer. More particularexamples of such cancers include multiple myeloma (MM), chronic lymphoidleukemia (CLL), mantle cell lymphoma (MCL), diffuse large B-celllymphoma (DLBCL), follicular lymphoma (FL), acute myeloid leukemia(AML), myelodysplastic syndrome (MDS), chronic myelogenous leukemia(CML), chronic myelomonocytic leukemia, acute promyelocytic leukemia(APL), chronic myeloproliferative disorder, thrombocytic leukemia,precursor B-cell acute lymphoblastic leukemia (pre-B-ALL), precursor Tcell acute lymphoblastic leukemia (pre-T-ALL), mast cell disease, mastcell leukemia, mast cell sarcoma, myeloid sarcomas, lymphoid leukemia,and undifferentiated leukemia. In some embodiments, the cancer is a Bcell cancer. In particular, cancer can include conditions involving theproduction of excess antibodies, such as monoclonal gammopathy, lightchain amyloidosis, monoclonal gammopathy of undetermined significanceand solitary plasmacytomas, isolated plasmacytoma and extramedullaryplasmacytoma.

“Tumor,” as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues. The terms “cancer,” “cancerous,” “cellproliferative disorder,” “proliferative disorder,” and “tumor” are notmutually exclusive as referred to herein.

“Effector functions” refer to those biological activities attributableto the Fc region of an antibody, which vary with the antibody isotype.Examples of antibody effector functions include: Clq binding andcomplement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cellactivation.

An “effective amount” of a compound, for example, an anti-FcRH5 antibodyof the invention or a composition (e.g., pharmaceutical composition)thereof, is at least the minimum amount required to achieve the desiredtherapeutic or prophylactic result, such as a measurable improvement orprevention of a particular disorder (e.g., a cell proliferativedisorder, e.g., cancer). An effective amount herein may vary accordingto factors such as the disease state, age, sex, and weight of thepatient, and the ability of the antibody to elicit a desired response inthe individual. An effective amount is also one in which any toxic ordetrimental effects of the treatment are outweighed by thetherapeutically beneficial effects. For prophylactic use, beneficial ordesired results include results such as eliminating or reducing therisk, lessening the severity, or delaying the onset of the disease,including biochemical, histological and/or behavioral symptoms of thedisease, its complications, and intermediate pathological phenotypespresenting during development of the disease. For therapeutic use,beneficial or desired results include clinical results such asdecreasing one or more symptoms resulting from the disease, increasingthe quality of life of those suffering from the disease, decreasing thedose of other medications required to treat the disease, enhancingeffect of another medication such as via targeting, delaying theprogression of the disease, and/or prolonging survival. In the case ofcancer or tumor, an effective amount of the drug may have the effect inreducing the number of cancer cells; reducing the tumor size; inhibiting(i.e., slow to some extent or desirably stop) cancer cell infiltrationinto peripheral organs; inhibit (i.e., slow to some extent and desirablystop) tumor metastasis; inhibiting to some extent tumor growth; and/orrelieving to some extent one or more of the symptoms associated with thedisorder. An effective amount can be administered in one or moreadministrations. For purposes of this invention, an effective amount ofdrug, compound, or pharmaceutical composition is an amount sufficient toaccomplish prophylactic or therapeutic treatment either directly orindirectly. As is understood in the clinical context, an effectiveamount of a drug, compound, or pharmaceutical composition may or may notbe achieved in conjunction with another drug, compound, orpharmaceutical composition. Thus, an “effective amount” may beconsidered in the context of administering one or more therapeuticagents, and a single agent may be considered to be given in an effectiveamount if, in conjunction with one or more other agents, a desirableresult may be or is achieved.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc regions andvariant Fc regions. In one embodiment, a human IgG heavy chain Fc regionextends from Cys226, or from Pro230, to the carboxyl-terminus of theheavy chain. However, the C-terminal lysine (Lys447) of the Fc regionmay or may not be present. Unless otherwise specified herein, numberingof amino acid residues in the Fc region or constant region is accordingto the EU numbering system, also called the EU index, as described inKabat et al. Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.,1991.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The term “FcRH5-positive cell” refers to a cell that expresses FcRH5 onits surface. In some embodiments, FcRH5 is one or more of FcRH5a,FcRH5b, FcRH5c, UniProt Identifier Q96RD9-2, and/or FcRH5d. In someembodiments, the FcRH5 is FcRH5c.

The term “FcRH5-positive cancer” refers to a cancer comprising cellsthat express FcRH5 on their surface. For the purposes of determiningwhether a cell expresses FcRH5 on the surface, FcRH5 mRNA expression isconsidered to correlate to FcRH5 expression on the cell surface. In someembodiments, expression of FcRH5 mRNA is determined by a method selectedfrom in situ hybridization and RT-PCR (including quantitative RT-PCR).Alternatively, expression of FcRH5 on the cell surface can bedetermined, for example, using antibodies to FcRH5 in a method such asimmunohistochemistry, FACS, etc. In some embodiments, FcRH5 is one ormore of FcRH5a, FcRH5b, FcRH5c, UniProt Identifier Q96RD9-2, and/orFcRH5d. In some embodiments, the FcRH5 is FcRH5c.

The terms “full-length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

The term “glycosylated forms of FcRH5” refers to naturally occurringforms of FcRH5 that are post-translationally modified by the addition ofcarbohydrate residues.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell either in vitro or in vivo.In one embodiment, growth inhibitory agent is growth inhibitory antibodythat prevents or reduces proliferation of a cell expressing an antigento which the antibody binds.

In another embodiment, the growth inhibitory agent may be one whichsignificantly reduces the percentage of cells in S phase. Examples ofgrowth inhibitory agents include agents that block cell cycleprogression (at a place other than S phase), such as agents that induceG1 arrest and M-phase arrest. Classical M-phase blockers include thevincas (vincristine and vinblastine), taxanes, and topoisomerase IIinhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, andbleomycin. Those agents that arrest G1 also spill over into S-phasearrest, for example, DNA alkylating agents such as tamoxifen,prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate,5-fluorouracil, and ara-C. Further information can be found inMendelsohn and Israel, eds., The Molecular Basis of Cancer, Chapter 1,entitled “Cell cycle regulation, oncogenes, and antineoplastic drugs” byMurakami et al. (W.B. Saunders, Philadelphia, 1995), e.g., p. 13. Thetaxanes (paclitaxel and docetaxel) are anticancer drugs both derivedfrom the yew tree. Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derivedfrom the European yew, is a semisynthetic analogue of paclitaxel(TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote theassembly of microtubules from tubulin dimers and stabilize microtubulesby preventing depolymerization, which results in the inhibition ofmitosis in cells.

The terms “host cell,” “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues. Human antibodies can beproduced using various techniques known in the art, includingphage-display libraries. Hoogenboom and Winter. J. Mol. Biol. 227:381,1991; Marks et al. J. Mol. Biol. 222:581, 1991. Also available for thepreparation of human monoclonal antibodies are methods described in Coleet al. Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985); Boerner et al. J. Immunol., 147(1):86-95, 1991. See also vanDijk and van de Winkel. Curr. Opin. Pharmacol. 5:368-74, 2001. Humanantibodies can be prepared by administering the antigen to a transgenicanimal that has been modified to produce such antibodies in response toantigenic challenge, but whose endogenous loci have been disabled, e.g.,immunized xenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584regarding XENOMOUSE™ technology). See also, for example, Li et al. Proc.Natl. Acad. Sci. USA. 103:3557-3562, 2006 regarding human antibodiesgenerated via a human B-cell hybridoma technology.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al. Sequences of Proteins of Immunological Interest, FifthEdition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal. supra. In one embodiment, for the VH, the subgroup is subgroup IIIas in Kabat et al. supra.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization.

The term “hypervariable region” or “HVR” as used herein refers to eachof the regions of an antibody variable domain which are hypervariable insequence (“complementarity determining regions” or “CDRs”) and/or formstructurally defined loops (“hypervariable loops”) and/or contain theantigen-contacting residues (“antigen contacts”). Generally, antibodiescomprise six HVRs: three in the VH (H1, H2, H3), and three in the VL(L1, L2, L3). Exemplary HVRs herein include:

(a) hypervariable loops occurring at amino acid residues 26-32 (L1),50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothiaand Lesk, J. Mol. Biol. 196:901-917, 1987);

(b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97(L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al. Sequencesof Proteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991));

(c) antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55(L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum etal. J. Mol. Biol. 262: 732-745, 1996); and

(d) combinations of (a), (b), and/or (c), including HVR amino acidresidues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1),26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3).

Unless otherwise indicated, HVR residues and other residues in thevariable domain (e.g., FR residues) are numbered herein according toKabat et al. supra.

An “immunoconjugate” is an antibody conjugated to one or moreheterologous molecule(s), including but not limited to a cytotoxicagent.

A “subject” or an “individual” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g., cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, thesubject or individual is a human.

An “isolated” antibody is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC). For review of methods for assessment of antibody purity, see,e.g., Flatman et al. J. Chromatogr. B 848:79-87, 2007.

An “isolated” nucleic acid refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

“Isolated nucleic acid encoding an anti-FcRH5 antibody” refers to one ormore nucleic acid molecules encoding antibody heavy and light chains (orfragments thereof), including such nucleic acid molecule(s) in a singlevector or separate vectors, and such nucleic acid molecule(s) present atone or more locations in a host cell.

“Isolated nucleic acid encoding an anti-CD3 antibody” refers to one ormore nucleic acid molecules encoding antibody heavy and light chains (orfragments thereof), including such nucleic acid molecule(s) in a singlevector or separate vectors, and such nucleic acid molecule(s) present atone or more locations in a host cell.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

A “naked antibody” refers to an antibody that is not conjugated to aheterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The nakedantibody may be present in a pharmaceutical formulation.

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150,000 daltons, composed oftwo identical light chains and two identical heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3).Similarly, from N- to C-terminus, each light chain has a variable region(VL), also called a variable light domain or a light chain variabledomain, followed by a constant light (CL) domain. The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (A), based on the amino acid sequence of its constant domain.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

The term “PD-1 axis binding antagonist” refers to a molecule thatinhibits the interaction of a PD-1 axis binding partner with either oneor more of its binding partner, so as to remove T cell dysfunctionresulting from signaling on the PD-1 signaling axis—with a result beingto restore or enhance T cell function (e.g., proliferation, cytokineproduction, target cell killing). As used herein, a PD-1 axis bindingantagonist includes a PD-1 binding antagonist, a PD-L1 bindingantagonist and a PD-L2 binding antagonist.

The term “PD-1 binding antagonist” refers to a molecule that decreases,blocks, inhibits, abrogates or interferes with signal transductionresulting from the interaction of PD-1 with one or more of its bindingpartners, such as PD-L1, PD-L2. In some embodiments, the PD-1 bindingantagonist is a molecule that inhibits the binding of PD-1 to one ormore of its binding partners. In a specific embodiment, the PD-1 bindingantagonist inhibits the binding of PD-1 to PD-L1 and/or PD-L2. Forexample, PD-1 binding antagonists include anti-PD-1 antibodies, antigenbinding fragments thereof, immunoadhesins, fusion proteins,oligopeptides and other molecules that decrease, block, inhibit,abrogate or interfere with signal transduction resulting from theinteraction of PD-1 with PD-L1 and/or PD-L2. In one embodiment, a PD-1binding antagonist reduces the negative co-stimulatory signal mediatedby or through cell surface proteins expressed on T lymphocytes mediatedsignaling through PD-1 so as render a dysfunctional T cell lessdysfunctional (e.g., enhancing effector responses to antigenrecognition). In some embodiments, the PD-1 binding antagonist is ananti-PD-1 antibody. In a specific embodiment, a PD-1 binding antagonistis MDX-1106 (nivolumab). In another specific embodiment, a PD-1 bindingantagonist is MK-3475 (pembrolizumab). In another specific embodiment, aPD-1 binding antagonist is CT-011 (pidilizumab). In another specificembodiment, a PD-1 binding antagonist is AMP-224. In another specificembodiment, a PD-1 binding antagonist is MED1-0680. In another specificembodiment, a PD-1 binding antagonist is PDR001. In another specificembodiment, a PD-1 binding antagonist is REGN2810. In another specificembodiment, a PD-1 binding antagonist is BGB-108.

The term “PD-L1 binding antagonist” refers to a molecule that decreases,blocks, inhibits, abrogates or interferes with signal transductionresulting from the interaction of PD-L1 with either one or more of itsbinding partners, such as PD-1, B7-1. In some embodiments, a PD-L1binding antagonist is a molecule that inhibits the binding of PD-L1 toits binding partners. In a specific embodiment, the PD-L1 bindingantagonist inhibits binding of PD-L1 to PD-1 and/or B7-1. In someembodiments, the PD-L1 binding antagonists include anti-PD-L1antibodies, antigen binding fragments thereof, immunoadhesins, fusionproteins, oligopeptides and other molecules that decrease, block,inhibit, abrogate or interfere with signal transduction resulting fromthe interaction of PD-L1 with one or more of its binding partners, suchas PD-1, B7-1. In one embodiment, a PD-L1 binding antagonist reduces thenegative co-stimulatory signal mediated by or through cell surfaceproteins expressed on T lymphocytes mediated signaling through PD-L1 soas to render a dysfunctional T cell less dysfunctional (e.g., enhancingeffector responses to antigen recognition). In some embodiments, a PD-L1binding antagonist is an anti-PD-L1 antibody. In still another specificembodiment, an anti-PD-L1 antibody is MPDL3280A (atezolizumab, marketedas TECENTRIQ™ with a WHO Drug Information (International NonproprietaryNames for Pharmaceutical Substances), Recommended INN: List 74, Vol. 29,No. 3, 2015 (see page 387)). In a specific embodiment, an anti-PD-L1antibody is YW243.55.S70. In another specific embodiment, an anti-PD-L1antibody is MDX-1105. In another specific embodiment, an anti PD-L1antibody is MSB0015718C. In still another specific embodiment, ananti-PD-L1 antibody is MED14736.

The term “PD-L2 binding antagonist” refers to a molecule that decreases,blocks, inhibits, abrogates or interferes with signal transductionresulting from the interaction of PD-L2 with either one or more of itsbinding partners, such as PD-1. In some embodiments, a PD-L2 bindingantagonist is a molecule that inhibits the binding of PD-L2 to one ormore of its binding partners. In a specific embodiment, the PD-L2binding antagonist inhibits binding of PD-L2 to PD-1. In someembodiments, the PD-L2 antagonists include anti-PD-L2 antibodies,antigen binding fragments thereof, immunoadhesins, fusion proteins,oligopeptides and other molecules that decrease, block, inhibit,abrogate or interfere with signal transduction resulting from theinteraction of PD-L2 with either one or more of its binding partners,such as PD-1. In one embodiment, a PD-L2 binding antagonist reduces thenegative co-stimulatory signal mediated by or through cell surfaceproteins expressed on T lymphocytes mediated signaling through PD-L2 soas render a dysfunctional T cell less dysfunctional (e.g., enhancingeffector responses to antigen recognition). In some embodiments, a PD-L2binding antagonist is an immunoadhesin.

The term “protein,” as used herein, refers to any native protein fromany vertebrate source, including mammals such as primates (e.g., humans)and rodents (e.g., mice and rats), unless otherwise indicated. The termencompasses “full-length,” unprocessed protein as well as any form ofthe protein that results from processing in the cell. The term alsoencompasses naturally occurring variants of the protein, e.g., splicevariants or allelic variants.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full-length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, antibodies ofthe invention are used to delay development of a disease or to slow theprogression of a disease.

As used herein, “delaying progression” of a disorder or disease means todefer, hinder, slow, retard, stabilize, and/or postpone development ofthe disease or disorder (e.g., a cell proliferative disorder, e.g.,cancer). This delay can be of varying lengths of time, depending on thehistory of the disease and/or individual being treated. As is evident toone skilled in the art, a sufficient or significant delay can, ineffect, encompass prevention, in that the individual does not developthe disease. For example, a late stage cancer, such as development ofmetastasis, may be delayed.

The term “epitope” refers to the particular site on an antigen moleculeto which an antibody binds. In some embodiments, the particular site onan antigen molecule to which an antibody binds is determined by hydroxylradical footprinting (e.g., FcRH5 binding domain). In some embodiments,the particular site on an antigen molecule to which an antibody binds isdetermined by crystallography.

By “reduce” or “inhibit” is meant the ability to cause an overalldecrease, for example, of 20% or greater, of 50% or greater, or of 75%,85%, 90%, 95%, or greater. In certain embodiments, reduce or inhibit canrefer to the effector function of an antibody that is mediated by theantibody Fc region, such effector functions specifically includingcomplement-dependent cytotoxicity (CDC), antibody-dependent cellularcytotoxicity (ADCC), and antibody-dependent celiular phagocytosis(ADCP).

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindtet al. Kuby Immunology, 6^(th) ed. W.H. Freeman and Co., page 91(2007).) A single VH or VL domain may be sufficient to conferantigen-binding specificity. Furthermore, antibodies that bind aparticular antigen may be isolated using a VH or VL domain from anantibody that binds the antigen to screen a library of complementary VLor VH domains, respectively. See, e.g., Portolano et al. J. Immunol.150:880-887, 1993; Clarkson et al. Nature 352:624-628, 1991.

A “variant Fc region” comprises an amino acid sequence which differsfrom that of a native sequence Fc region by virtue of at least one aminoacid modification, preferably one or more amino acid substitution(s).Preferably, the variant Fc region has at least one amino acidsubstitution compared to a native sequence Fc region or to the Fc regionof a parent polypeptide, e.g., from about one to about ten amino acidsubstitutions, and preferably from about one to about five amino acidsubstitutions in a native sequence Fc region or in the Fc region of theparent polypeptide. The variant Fc region herein will preferably possessat least about 80% homology with a native sequence Fc region and/or withan Fc region of a parent polypeptide, and most preferably at least about90% homology therewith, more preferably at least about 95% homologytherewith.

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors.”

As used herein, “administering” is meant a method of giving a dosage ofa compound (e.g., an anti-FcRH5 antibody of the invention or a nucleicacid encoding an anti-FcRH5 antibody of the invention) or a composition(e.g., a pharmaceutical composition, e.g., a pharmaceutical compositionincluding an anti-FcRH5 antibody of the invention) to a subject. Thecompositions utilized in the methods described herein can beadministered, for example, intramuscularly, intravenously,intradermally, percutaneously, intraarterially, intraperitoneally,intralesionally, intracranially, intraarticularly, intraprostatically,intrapleurally, intratracheally, intranasally, intravitreally,intravaginally, intrarectally, topically, intratumorally, peritoneally,subcutaneously, subconjunctivally, intravesicularlly, mucosally,intrapericardially, intraumbilically, intraocularly, orally, topically,locally, by inhalation, by injection, by infusion, by continuousinfusion, by localized perfusion bathing target cells directly, bycatheter, by lavage, in cremes, or in lipid compositions. The method ofadministration can vary depending on various factors (e.g., the compoundor composition being administered and the severity of the condition,disease, or disorder being treated).

II. Compositions and Methods

In one aspect, the invention is based in part on anti-FcRH5 antibodies.In certain embodiments, the anti-FcRH5 antibodies are multispecific(e.g., bispecific) and bind, in addition to FcRH5 or a fragment thereof,a second biological molecule (e.g., a cell surface antigen, e.g., a Tcell marker, e.g., CD3 (e.g., CD3ε and/or CD3γ)). Antibodies of theinvention are useful, for example, for diagnosing and/or treating ordelaying the progression of a cell proliferative disorder (e.g., cancer,e.g., an FcRH5-positive cancer, e.g., multiple myeloma) in a subject.

A. Exemplary Anti-FcRH5 Antibodies

In one aspect, the invention provides an anti-FcRH5 antibody having abinding domain comprising at least one, two, three, four, five, or sixhypervariable regions (HVRs) selected from (a) an HVR-H1 comprising theamino acid sequence of SEQ ID NO: 1; (b) an HVR-H2 comprising the aminoacid sequence of SEQ ID NO: 2; (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 3; (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 4; (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 5; and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 6.

In some instances, the invention provides an anti-FcRH5 antibody havinga binding domain comprising at least one, two, three, four, five, or sixhypervariable regions (HVRs) selected from (a) an HVR-H1 comprising theamino acid sequence of SEQ ID NO: 1; (b) an HVR-H2 comprising the aminoacid sequence of SEQ ID NO: 8; (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 9; (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 12; (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 16; and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 23. In some instances, the anti-FcRH5 antibodymay have a heavy chain variable (VH) domain including an amino acidsequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%)sequence identity to, or the sequence of, SEQ ID NO: 104 and/or a lightchain variable (VL) domain comprising an amino acid sequence having atleast 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to, orthe sequence of, SEQ ID NO: 105. In some instances, the anti-FcRH5antibody comprises at least one (e.g., 1, 2, 3, or 4) of heavy chainframework regions FR-H1, FR-H2, FR-H3, and FR-H4 comprising thesequences of SEQ ID NOs: 52, 54, 46, and 47, respectively. In someinstances, the anti-FcRH5 antibody may have a heavy chain variable (VH)domain including the amino acid sequence of SEQ ID NO: 104. In someinstances, the anti-FcRH5 antibody further includes at least one (e.g.,1, 2, 3, or 4) of the light chain framework regions FR-L1, FR-L2, FR-L3,and FR-L4 comprising the sequences of SEQ ID NOs: 48, 57, 50, and 51,respectively. In some instances, the anti-FcRH5 antibody may have a VLdomain comprising the amino acid sequence of SEQ ID NO: 105. In aparticular instance, the anti-FcRH5 antibody can be 1G7.v85, or aderivative or clonal relative thereof. In some instances, for example,the invention provides an anti-FcRH5 antibody having a binding domaincomprising (a) a VH domain comprising an amino acid sequence of SEQ IDNO: 104 and (b) a VL domain comprising an amino acid sequence of SEQ IDNO: 105.

In some instances, the invention provides an anti-FcRH5 antibody havinga binding domain comprising at least one, two, three, four, five, or sixhypervariable regions (HVRs) selected from SEQ ID NO: 1; (b) an HVR-H2comprising the amino acid sequence of SEQ ID NO: 8; (c) an HVR-H3comprising the amino acid sequence of SEQ ID NO: 10; (d) an HVR-L1comprising the amino acid sequence of SEQ ID NO: 14; (e) an HVR-L2comprising the amino acid sequence of SEQ ID NO: 16; and (f) an HVR-L3comprising the amino acid sequence of SEQ ID NO: 23. In some instances,the anti-FcRH5 antibody may have a VH domain including an amino acidsequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%)sequence identity to, or the sequence of, SEQ ID NO: 106 and/or a VLdomain comprising an amino acid sequence having at least 80% (e.g., 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99%) sequence identity to, or the sequence of,SEQ ID NO: 107. In some instances, the anti-FcRH5 antibody comprises atleast one (e.g., 1, 2, 3, or 4) of heavy chain framework regions FR-H1,FR-H2, FR-H3, and FR-H4 comprising the sequences of SEQ ID NO: 53, 54,46, and 47, respectively. In some instances, the anti-FcRH5 antibody mayhave a VH domain comprising the amino acid sequence of SEQ ID NO: 106.In some instances, the anti-FcRH5 further includes at least one (e.g.,1, 2, 3, or 4) of the light chain framework regions FR-L1, FR-L2, FR-L3,and FR-L4 comprising the sequences of SEQ ID NOs: 48, 57, 50, and 51,respectively. In some instances, the anti-FcRH5 antibody may have a VLdomain comprising the amino acid sequence of SEQ ID NO: 107. In aparticular instance, the anti-FcRH5 antibody can be 1G7.v93, or aderivative or clonal relative thereof. In some instances, for example,the invention provides an anti-FcRH5 antibody having a binding domaincomprising (a) a VH domain comprising an amino acid sequence of SEQ IDNO: 106 and (b) a VL domain comprising an amino acid sequence of SEQ IDNO: 107.

In some instances, the invention provides an anti-FcRH5 antibody havinga binding domain comprising at least one, two, three, four, five, or sixhypervariable regions (HVRs) selected from (a) an HVR-H1 comprising theamino acid sequence of SEQ ID NO: 1; (b) an HVR-H2 comprising the aminoacid sequence of SEQ ID NO: 7; (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 9; (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 11; (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 15; and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 20. In some instances, the anti-FcRH5 antibody ofclaim may have a VH domain comprising an amino acid sequence having atleast 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to, orthe sequence of, SEQ ID NO: 82 and/or a VL domain comprising an aminoacid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%)sequence identity to, or the sequence of, SEQ ID NO: 83. In someinstances, the anti-FcRH5 antibody comprises at least one (e.g., 1, 2,3, or 4) of heavy chain framework regions FR-H1, FR-H2, FR-H3, and FR-H4comprising the sequences of SEQ ID NO: 52, 54, 46, and 47. In someinstances, the anti-FcRH5 antibody may have a VH domain comprising theamino acid sequence of SEQ ID NO: 82. In some instances, the anti-FcRH5antibody further includes at least one (e.g., 1, 2, 3, or 4) of thelight chain framework regions FR-L1, FR-L2, FR-L3, and FR-L4 comprisingthe sequences of SEQ ID NOs: 48, 56, 50, and 51. In some instances, theanti-FcRH5 antibody may have a VL domain comprising the amino acidsequence of SEQ ID NO: 83. In a particular instance, the anti-FcRH5antibody can be 1G7.v1, or a derivative or clonal relative thereof. Insome instances, for example, the invention provides an anti-FcRH5antibody having a binding domain comprising (a) a VH domain comprisingan amino acid sequence of SEQ ID NO: 82 and (b) a VL domain comprisingan amino acid sequence of SEQ ID NO: 83.

In some instances, the anti-FcRH5 antibody includes a binding domaincomprising at least one, two, three, four, five, or six hypervariableregions (HVRs) selected from (a) an HVR-H1 comprising the amino acidsequence of SEQ ID NO: 1; (b) an HVR-H2 comprising the amino acidsequence of SEQ ID NO: 7; (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 9; (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 12; (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 16; and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 21. In some instances, the anti-FcRH5 antibodyincludes a VH domain comprising an amino acid sequence having at least80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 84 and/or a VL domain comprising an amino acidsequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%)sequence identity to, or the sequence of, SEQ ID NO: 85. In someinstances, the anti-FcRH5 antibody further includes the heavy chainframework regions FR-H1, FR-H2, FR-H3, and FR-H4 of SEQ ID NO: 52, 54,46, and 47, respectively. In some instances, the anti-FcRH5 antibody mayhave a VH domain comprising the amino acid sequence of SEQ ID NO: 84. Insome instances, the anti-FcRH5 antibody further includes at least one(e.g., 1, 2, 3, or 4) of the light chain framework regions FR-L1, FR-L2,FR-L3, and FR-L4 comprising the sequences of SEQ ID NOs: 48, 57, 50, and51, respectively. In some instances, the anti-FcRH5 antibody may have aVL domain comprising the amino acid sequence of SEQ ID NO: 85. In aparticular instance, the anti-FcRH5 antibody can be 1G7.v1.1, or aderivative or clonal relative thereof. In some instances, for example,the invention provides an anti-FcRH5 antibody having a binding domaincomprising (a) a VH domain comprising an amino acid sequence of SEQ IDNO: 84 and (b) a VL domain comprising an amino acid sequence of SEQ IDNO: 85.

In some instances, the anti-FcRH5 antibody has a binding domaincomprising six hypervariable regions (HVRs) (a) an HVR-H1 comprising theamino acid sequence of SEQ ID NO: 1; (b) an HVR-H2 comprising the aminoacid sequence of SEQ ID NO: 7; (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 9; (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 12; (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 17; and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 22. In some instances, the anti-FcRH5 antibodyincludes a VH domain comprising an amino acid sequence having at least80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 86 and/or a VL domain comprising an amino acidsequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%)sequence identity to, or the sequence of, SEQ ID NO: 87. In someinstances, the anti-FcRH5 antibody further comprises further includesthe heavy chain framework regions FR-H1, FR-H2, FR-H3, and FR-H4comprising the amino acid sequences of SEQ ID NO: 52, 54, 46, and 47. Insome instances, the anti-FcRH5 antibody may have a VH domain comprisingthe amino acid sequence of SEQ ID NO: 86. In some instances, theanti-FcRH5 antibody further includes at least one (e.g., 1, 2, 3, or 4)of the light chain framework regions FR-L1, FR-L2, FR-L3, and FR-L4comprising the sequences of SEQ ID NOs: 48, 57, 50, and 51,respectively. In some instances, the anti-FcRH5 antibody of claim mayhave a VL domain comprising the amino acid sequence of SEQ ID NO: 87. Ina particular instance, the anti-FcRH5 antibody can be 1G7.v1.2, or aderivative or clonal relative thereof. In some instances, for example,the invention provides an anti-FcRH5 antibody having a binding domaincomprising (a) a VH domain comprising an amino acid sequence of SEQ IDNO: 86 and (b) a VL domain comprising an amino acid sequence of SEQ IDNO: 87.

In some instances, the anti-FcRH5 antibody having a binding domaincomprising six hypervariable regions (HVRs) (a) an HVR-H1 comprising theamino acid sequence of SEQ ID NO: 1; (b) an HVR-H2 comprising the aminoacid sequence of SEQ ID NO: 7; (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 9; (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 13; (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 16; and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 21. In some instances, the anti-FcRH5 antibodycomprises a VH domain comprising an amino acid sequence having at least80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 88 and/or a VL domain comprising an amino acidsequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%)sequence identity to, or the sequence of, SEQ ID NO: 89. In someinstances, the anti-FcRH5 antibody further includes the heavy chainframework regions FR-H1, FR-H2, FR-H3, and FR-H4 comprising the aminoacid sequences of SEQ ID NOs: 52, 54, 46, and 47, respectively. In someinstances, the anti-FcRH5 antibody may have a VH domain comprising theamino acid sequence of SEQ ID NO: 88. In some instances, the anti-FcRH5antibody further includes at least one (e.g., 1, 2, 3, or 4) of thelight chain framework regions FR-L1, FR-L2, FR-L3, and FR-L4 comprisingthe sequences of SEQ ID NOs: 48, 57, 50, and 51, respectively. In someinstances, the anti-FcRH5 antibody may have a VL domain comprising theamino acid sequence of SEQ ID NO: 89. In a particular instance, theanti-FcRH5 antibody can be 1G7.v1.3, or a derivative or clonal relativethereof. In some instances, for example, the invention provides ananti-FcRH5 antibody comprising a binding domain comprising (a) a VHdomain comprising an amino acid sequence of SEQ ID NO: 88 and (b) a VLdomain comprising an amino acid sequence of SEQ ID NO: 89.

In some instances, the anti-FcRH5 antibody includes a binding domaincomprising at least one, two, three, four, five, or six hypervariableregions (HVRs) selected from (a) an HVR-H1 comprising the amino acidsequence of SEQ ID NO: 1; (b) an HVR-H2 comprising the amino acidsequence of SEQ ID NO: 7; (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 9; (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 12; (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 16; and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 23. In some instances, the anti-FcRH5 antibodymay have a binding domain including a VH domain comprising an amino acidsequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%)sequence identity to, or the sequence of, SEQ ID NO: 90 and/or a VLdomain comprising an amino acid sequence having at least 80% (e.g., 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99%) sequence identity to, or the sequence of,SEQ ID NO: 91. In some instances, the anti-FcRH5 antibody furtherincludes the heavy chain framework regions FR-H1, FR-H2, FR-H3, andFR-H4 SEQ ID NO: 52, 54, 46, and 47. In some instances, the anti-FcRH5antibody may have a VH domain comprising the amino acid sequence of SEQID NO: 90. In some instances, the anti-FcRH5 antibody further includesat least one (e.g., 1, 2, 3, or 4) of the light chain framework regionsFR-L1, FR-L2, FR-L3, and FR-L4 comprising the sequences of SEQ ID NOs:48, 57, 50, and 51, respectively. In some instances, the anti-FcRH5antibody may have a VL domain comprising the amino acid sequence of SEQID NO: 91. In a particular instance, the anti-FcRH5 antibody can be1G7.v1.4, or a derivative or clonal relative thereof. In some instances,for example, the invention provides an anti-FcRH5 antibody having abinding domain comprising (a) a VH domain comprising an amino acidsequence of SEQ ID NO: 90 and (b) a VL domain comprising an amino acidsequence of SEQ ID NO: 91.

In some instances, the anti-FcRH5 antibody includes a binding domaincomprising at least one, two, three, four, five, or six hypervariableregions (HVRs) selected from (a) an HVR-H1 comprising the amino acidsequence of SEQ ID NO: 1; (b) an HVR-H2 comprising the amino acidsequence of SEQ ID NO: 7; (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 9; (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 11; (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 18; and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 22. In some instances, the anti-FcRH5 antibodyincludes a VH domain comprising an amino acid sequence having at least80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 92 and/or a VL domain comprising an amino acidsequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%)sequence identity to, or the sequence of, SEQ ID NO: 93. In someinstances, the anti-FcRH5 antibody comprises at least one (e.g., 1, 2,3, or 4) of heavy chain framework regions FR-H1, FR-H2, FR-H3, and FR-H4comprising the sequences of SEQ ID NOs: 52, 54, 46, and 47,respectively. In some instances, the anti-FcRH5 antibody may have a VHdomain comprising the amino acid sequence of SEQ ID NO: 92. In someinstances, the anti-FcRH5 antibody further includes at least one (e.g.,1, 2, 3, or 4) of the light chain framework regions FR-L1, FR-L2, FR-L3,and FR-L4 comprising the sequences of SEQ ID NOs: 48, 56, 50, and 51,respectively. In some instances, the anti-FcRH5 antibody may have a VLdomain comprising the amino acid sequence of SEQ ID NO: 93. In aparticular instance, the anti-FcRH5 antibody can be 1G7.v1.5, or aderivative or clonal relative thereof. In some instances, for example,the invention provides an anti-FcRH5 antibody having a binding domaincomprising (a) a VH domain comprising an amino acid sequence of SEQ IDNO: 92 and (b) a VL domain comprising an amino acid sequence of SEQ IDNO: 93.

In some instances, the anti-FcRH5 antibody includes a binding domaincomprising at least one, two, three, four, five, or six hypervariableregions (HVRs) selected from (a) an HVR-H1 comprising the amino acidsequence of SEQ ID NO: 1; (b) an HVR-H2 comprising the amino acidsequence of SEQ ID NO: 7; (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 9; (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 11; (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 19; and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 24. In some instances. The anti-FcRH5 antibodyincludes a VH domain comprising an amino acid sequence having at least80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 94 and/or a VL domain comprising an amino acidsequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%)sequence identity to, or the sequence of, SEQ ID NO: 95. In someinstances, the anti-FcRH5 antibody includes the heavy chain frameworkregions FR-H1, FR-H2, FR-H3, and FR-H4 comprising the amino acidsequences of SEQ ID NOs: 52, 54, 46, and 47, respectively. In someinstances, the anti-FcRH5 antibody may have a VH domain comprising theamino acid sequence of SEQ ID NO: 94. In some instances, the anti-FcRH5antibody further includes at least one (e.g., 1, 2, 3, or 4) of thelight chain framework regions FR-L1, FR-L2, FR-L3, and FR-L4 comprisingthe sequences of SEQ ID NOs: 48, 57, 50, and 51, respectively. In someinstances, the anti-FcRH5 antibody includes a VL domain comprising theamino acid sequence of SEQ ID NO: 95. In a particular instance, theanti-FcRH5 antibody can be 1G7.v1.6, or a derivative or clonal relativethereof. In some instances, for example, the invention provides ananti-FcRH5 antibody having a binding domain including (a) a VH domaincomprising an amino acid sequence of SEQ ID NO: 94 and (b) a VL domaincomprising an amino acid sequence of SEQ ID NO: 95.

In some instances, the anti-FcRH5 antibody includes a binding domaincomprising at least one, two, three, four, five, or six hypervariableregions (HVRs) selected from (a) an HVR-H1 comprising the amino acidsequence of SEQ ID NO: 1; (b) an HVR-H2 comprising the amino acidsequence of SEQ ID NO: 7; (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 9; (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 12; (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 18; and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 25. In some instances, the anti-FcRH5 antibodyincludes a VH domain comprising an amino acid sequence having at least80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 96 and/or a VL domain comprising an amino acidsequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%)sequence identity to, or the sequence of, SEQ ID NO: 97. In someinstances, the anti-FcRH5 antibody comprises at least one (e.g., 1, 2,3, or 4) of heavy chain framework regions FR-H1, FR-H2, FR-H3, and FR-H4comprising the sequences of SEQ ID NOs: 53, 54, 46, and 47. In someinstances, the anti-FcRH5 antibody includes a VH domain comprising theamino acid sequence of SEQ ID NO: 96. In some instances, the anti-FcRH5includes at least one (e.g., 1, 2, 3, or 4) of the light chain frameworkregions FR-L1, FR-L2, FR-L3, and FR-L4 comprising the sequences of SEQID NOs: 48, 57, 50, and 51. In some instances, the anti-FcRH5 antibodyincludes a VL domain comprising the amino acid sequence of SEQ ID NO:97. In a particular instance, the anti-FcRH5 antibody can be 1G7.v1.7,or a derivative or clonal relative thereof. In some instances, forexample, the invention provides an anti-FcRH5 antibody, having a bindingdomain including (a) a VH domain comprising an amino acid sequence ofSEQ ID NO: 96 and (b) a VL domain comprising an amino acid sequence ofSEQ ID NO: 97.

In some instances, the anti-FcRH5 antibody includes a binding domaincomprising at least one, two, three, four, five, or six hypervariableregions (HVRs) selected from (a) an HVR-H1 comprising the amino acidsequence of SEQ ID NO: 1; (b) an HVR-H2 comprising the amino acidsequence of SEQ ID NO: 7; (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 9; (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 12; (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 18; and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 25. In some instances, the anti-FcRH5 antibodyincludes a VH domain comprising an amino acid sequence having at least80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 98, and/or a VL domain comprising an amino acidsequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%)sequence identity to, or the sequence of, SEQ ID NO: 99. In someinstances, the anti-FcRH5 comprises at least one (e.g., 1, 2, 3, or 4)of heavy chain framework regions FR-H1, FR-H2, FR-H3, and FR-H4comprising the sequences of SEQ ID NOs: 52, 55, 46, and 47,respectively. In some instances, the anti-FcRH5 antibody includes a VHdomain comprising the amino acid sequence of SEQ ID NO: 98. In someinstances, the anti-FcRH5 antibody includes at least one (e.g., 1, 2, 3,or 4) of the light chain framework regions FR-L1, FR-L2, FR-L3, andFR-L4 comprising the sequences of SEQ ID NOs: 48, 57, 50, and 51. Insome instances, the anti-FcRH5 antibody may have a VL domain comprisingthe amino acid sequence of SEQ ID NO: 99. In a particular instance, theanti-FcRH5 antibody can be 1G7.v1.13, or a derivative or clonal relativethereof. In some instances, for example, an anti-FcRH5 antibody mayinclude a VH domain comprising an amino acid sequence of SEQ ID NO: 98and (b) a VL domain comprising an amino acid sequence of SEQ ID NO: 99.

In some instances, the anti-FcRH5 antibody includes a binding domaincomprising at least one, two, three, four, five, or six hypervariableregions (HVRs) selected from (a) an HVR-H1 comprising the amino acidsequence of SEQ ID NO: 1; (b) an HVR-H2 comprising the amino acidsequence of SEQ ID NO: 7; (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 9; (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 12; (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 18; and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 25. In some instances, the anti-FcRH5 antibodyincludes (a) a VH domain comprising an amino acid sequence having atleast 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to, orthe sequence of, SEQ ID NO: 100 and/or a VL domain comprising an aminoacid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%)sequence identity to, or the sequence of, SEQ ID NO: 101. In someinstances, the anti-FcRH5 antibody comprises at least one (e.g., 1, 2,3, or 4) of heavy chain framework regions FR-H1, FR-H2, FR-H3, and FR-H4comprising the sequences of SEQ ID NOs: 52, 54, 46, and 47. In someinstances, the anti-FcRH5 may have a VH domain comprising the amino acidsequence of SEQ ID NO: 100. In some instances, The anti-FcRH5 antibodyof any one of claims 79-82, wherein the antibody further includes atleast one (e.g., 1, 2, 3, or 4) of the light chain framework regionsFR-L1, FR-L2, FR-L3, and FR-L4 comprising the sequences of SEQ ID NOs:48, 57, 50, and 51. In some instances, the anti-FcRH5 antibody may havea VL domain comprising the amino acid sequence of SEQ ID NO: 101. In aparticular instance, the anti-FcRH5 antibody can be 1G7.v1.13.1, or aderivative or clonal relative thereof. In some instances, for example,the invention provides an anti-FcRH5 antibody including (a) a VH domaincomprising an amino acid sequence of SEQ ID NO: 100 and (b) a VL domaincomprising an amino acid sequence of SEQ ID NO: 101.

In some instances, the anti-FcRH5 antibody includes a binding domaincomprising at least one, two, three, four, five, or six hypervariableregions (HVRs) selected from (a) an HVR-H1 comprising the amino acidsequence of SEQ ID NO: 1; (b) an HVR-H2 comprising the amino acidsequence of SEQ ID NO: 8; (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 9; (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 11; (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 15; and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 20. In some instances, the anti-FcRH5 antibodyincludes (a) a VH domain comprising an amino acid sequence having atleast 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to, orthe sequence of, SEQ ID NO: 102 and/or (b) a VL domain comprising anamino acid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99%) sequence identity to, or the sequence of, SEQ ID NO: 103. In someinstances, the anti-FcRH5 antibody further comprises at least one (e.g.,1, 2, 3, or 4) of heavy chain framework regions FR-H1, FR-H2, FR-H3, andFR-H4 comprising the sequences of SEQ ID NOs: 52, 54, 46, and 47,respectively. In some instances, the anti-FcRH5 antibody may have a VHdomain comprising the amino acid sequence of SEQ ID NO: 102. In someinstances, the anti-FcRH5 antibody further includes at least one (e.g.,1, 2, 3, or 4) of the light chain framework regions FR-L1, FR-L2, FR-L3,and FR-L4 comprising the sequences of SEQ ID NOs: 48, 56, 50, and 51. Insome instances, the anti-FcRH5 antibody may have a VL domain comprisingthe amino acid sequence of SEQ ID NO: 103. In a particular instance, theanti-FcRH5 antibody can be 1G7.v87, or a derivative or clonal relativethereof. In some instances, for example, the invention provides ananti-FcRH5 antibody comprising (a) a VH domain comprising an amino acidsequence of SEQ ID NO: 102 and (b) a VL domain comprising an amino acidsequence of SEQ ID NO: 103.

In some instances, an anti-FcRH5 antibody may have a binding domaincomprising at least one, two, three, four, five, or six hypervariableregions (HVRs) selected from (a) an HVR-H1 comprising the amino acidsequence of SEQ ID NO: 32; (b) an HVR-H2 comprising the amino acidsequence of SEQ ID NO: 33; (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 34; (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 35; (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 36; and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 37. In some instances, the anti-FcRH5 comprises aVH domain comprising an amino acid sequence having at least 80% (e.g.,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to, or the sequenceof, SEQ ID NO: 110 and/or a VL domain comprising an amino acid sequencehaving at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequenceidentity to, or the sequence of, SEQ ID NO: 111. In some instances, theanti-FcRH5 antibody comprises at least one (e.g., 1, 2, 3, or 4) ofheavy chain framework regions FR-H1, FR-H2, FR-H3, and FR-H4 comprisingthe sequences of SEQ ID NOs: 66, 67, 68, and 69, respectively. In someinstances, the anti-FcRH5 antibody of claim 95, wherein the VH domaincomprising the amino acid sequence of SEQ ID NO: 110. In some instances,the anti-FcRH5 antibody further includes at least one (e.g., 1, 2, 3, or4) of the light chain framework regions FR-L1, FR-L2, FR-L3, and FR-L4comprising the sequences of SEQ ID NOs: 70, 71, 72, and 73. In someinstances, the anti-FcRH5 antibody may have a VL domain comprising theamino acid sequence of SEQ ID NO: 111. In a particular instance, theanti-FcRH5 antibody can be 17B1, or a derivative or clonal relativethereof. In some instances, for example, the invention provides ananti-FcRH5 antibody comprising (a) a VH domain comprising an amino acidsequence of SEQ ID NO: 110 and (b) a VL domain comprising an amino acidsequence of SEQ ID NO: 111.

In some instances, an anti-FcRH5 antibody having a binding domaincomprising at least one, two, three, four, five, or six hypervariableregions (HVRs) selected from (a) an HVR-H1 comprising the amino acidsequence of SEQ ID NO: 38; (b) an HVR-H2 comprising the amino acidsequence of SEQ ID NO: 39; (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 40; (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 41; (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 42; and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 43. In some instances, the anti-FcRH5 antibodyincludes a VH domain comprising an amino acid sequence having at least80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 112 and/or a VL domain comprising an amino acidsequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%)sequence identity to, or the sequence of, SEQ ID NO: 113. In someinstances, the anti-FcRH5 comprises at least one (e.g., 1, 2, 3, or 4)of heavy chain framework regions FR-H1, FR-H2, FR-H3, and FR-H4comprising the sequences of SEQ ID NO: 74, 75, 76, and 77, respectively.In some instances, the anti-FcRH5 antibody may have a VH domaincomprising the amino acid sequence of SEQ ID NO: 112. In some instances,the anti-FcRH5 antibody further includes at least one (e.g., 1, 2, 3, or4) of the light chain framework regions FR-L1, FR-L2, FR-L3, and FR-L4comprising the sequences of SEQ ID NOs: 78, 79, 80, and 81. In someinstances, the anti-FcRH5 antibody may have a VL domain comprising theamino acid sequence of SEQ ID NO: 113. In a particular instance, theanti-FcRH5 antibody can be 15G8, or a derivative or clonal relativethereof. In some instances, for example, the invention provides ananti-FcRH5 antibody comprising (a) a VH domain comprising an amino acidsequence of SEQ ID NO: 112 and (b) a VL domain comprising an amino acidsequence of SEQ ID NO: 113.

In some instances, the invention provides an anti-FcRH5 antibody havinga binding domain comprising at least one, two, three, four, five, or sixhypervariable regions (HVRs) selected from (a) an HVR-H1 comprising theamino acid sequence of SEQ ID NO: 26; (b) an HVR-H2 comprising the aminoacid sequence of SEQ ID NO: 27; (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 28; (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 29; (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 30; and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 31. In some instances, the anti-FcRH5 antibodyincludes a VH domain comprising an amino acid sequence having at least80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 108 and/or a VL domain comprising an amino acidsequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%)sequence identity to, or the sequence of, SEQ ID NO: 109. In someinstances, the anti-FcRH5 antibody comprises at least one (e.g., 1, 2,3, or 4) of heavy chain framework regions FR-H1, FR-H2, FR-H3, and FR-H4comprising the sequences of SEQ ID NOs: 58, 59, 60, and 61,respectively. In some instances, the anti-FcRH5 antibody may have a VHdomain comprising the amino acid sequence of SEQ ID NO: 108. In someinstances, the anti-FcRH5 antibody further includes at least one (e.g.,1, 2, 3, or 4) of the light chain framework regions FR-L1, FR-L2, FR-L3,and FR-L4 comprising the sequences of SEQ ID NOs: 62, 63, 64, and 65. Insome instances, the anti-FcRH5 antibody may have a VL domain comprisingthe amino acid sequence of SEQ ID NO: 109. In a particular instance, theanti-FcRH5 antibody can be 7D8, or a derivative or clonal relativethereof. In some instances, for example, the invention provides ananti-FcRH5 antibody comprising (a) a VH domain comprising an amino acidsequence of SEQ ID NO: 108 and (b) a VL domain comprising an amino acidsequence of SEQ ID NO: 109.

In some instances, the invention provides an anti-FcRH5 antibody havinga binding domain comprising at least one, two, three, four, five, or sixhypervariable regions (HVRs) selected from (a) an HVR-H1 comprising theamino acid sequence of SEQ ID NO: 1; (b) an HVR-H2 comprising the aminoacid sequence of SEQ ID NO: 7; (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 9; (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 11; (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 15; and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 20. In some instances, the anti-FcRH5 antibody ofclaim may have a VH domain comprising an amino acid sequence having atleast 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to, orthe sequence of, SEQ ID NO: 185 and/or a VL domain comprising an aminoacid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%)sequence identity to, or the sequence of, SEQ ID NO: 186. In someinstances, the anti-FcRH5 antibody comprises at least one (e.g., 1, 2,3, or 4) of heavy chain framework regions FR-H1, FR-H2, FR-H3, and FR-H4comprising the sequences of SEQ ID NO: 179, 54, 46, and 47. In someinstances, the anti-FcRH5 antibody may have a VH domain comprising theamino acid sequence of SEQ ID NO: 185. In some instances, the anti-FcRH5antibody further includes at least one (e.g., 1, 2, 3, or 4) of thelight chain framework regions FR-L1, FR-L2, FR-L3, and FR-L4 comprisingthe sequences of SEQ ID NOs: 48, 56, 50, and 51. In some instances, theanti-FcRH5 antibody may have a VL domain comprising the amino acidsequence of SEQ ID NO: 186. In a particular instance, the anti-FcRH5antibody can be 1G7.v1A, or a derivative or clonal relative thereof. Insome instances, for example, the invention provides an anti-FcRH5antibody having a binding domain comprising (a) a VH domain comprisingan amino acid sequence of SEQ ID NO: 185 and (b) a VL domain comprisingan amino acid sequence of SEQ ID NO: 186.

In some instances, the invention provides an anti-FcRH5 antibody havinga binding domain comprising at least one, two, three, four, five, or sixhypervariable regions (HVRs) selected from (a) an HVR-H1 comprising theamino acid sequence of SEQ ID NO: 1; (b) an HVR-H2 comprising the aminoacid sequence of SEQ ID NO: 7; (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 9; (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 11; (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 15; and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 20. In some instances, the anti-FcRH5 antibody ofclaim may have a VH domain comprising an amino acid sequence having atleast 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to, orthe sequence of, SEQ ID NO: 187 and/or a VL domain comprising an aminoacid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%)sequence identity to, or the sequence of, SEQ ID NO: 188. In someinstances, the anti-FcRH5 antibody comprises at least one (e.g., 1, 2,3, or 4) of heavy chain framework regions FR-H1, FR-H2, FR-H3, and FR-H4comprising the sequences of SEQ ID NO: 180, 54, 46, and 47. In someinstances, the anti-FcRH5 antibody may have a VH domain comprising theamino acid sequence of SEQ ID NO: 187. In some instances, the anti-FcRH5antibody further includes at least one (e.g., 1, 2, 3, or 4) of thelight chain framework regions FR-L1, FR-L2, FR-L3, and FR-L4 comprisingthe sequences of SEQ ID NOs: 48, 56, 50, and 51. In some instances, theanti-FcRH5 antibody may have a VL domain comprising the amino acidsequence of SEQ ID NO: 188. In a particular instance, the anti-FcRH5antibody can be 1G7.v1B, or a derivative or clonal relative thereof. Insome instances, for example, the invention provides an anti-FcRH5antibody having a binding domain comprising (a) a VH domain comprisingan amino acid sequence of SEQ ID NO: 187 and (b) a VL domain comprisingan amino acid sequence of SEQ ID NO: 188.

In some instances, the invention provides an anti-FcRH5 antibody havinga binding domain comprising at least one, two, three, four, five, or sixhypervariable regions (HVRs) selected from (a) an HVR-H1 comprising theamino acid sequence of SEQ ID NO: 1; (b) an HVR-H2 comprising the aminoacid sequence of SEQ ID NO: 7; (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 9; (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 11; (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 15; and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 20. In some instances, the anti-FcRH5 antibody ofclaim may have a VH domain comprising an amino acid sequence having atleast 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to, orthe sequence of, SEQ ID NO: 189 and/or a VL domain comprising an aminoacid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%)sequence identity to, or the sequence of, SEQ ID NO: 190. In someinstances, the anti-FcRH5 antibody comprises at least one (e.g., 1, 2,3, or 4) of heavy chain framework regions FR-H1, FR-H2, FR-H3, and FR-H4comprising the sequences of SEQ ID NO: 181, 54, 46, and 47. In someinstances, the anti-FcRH5 antibody may have a VH domain comprising theamino acid sequence of SEQ ID NO: 189. In some instances, the anti-FcRH5antibody further includes at least one (e.g., 1, 2, 3, or 4) of thelight chain framework regions FR-L1, FR-L2, FR-L3, and FR-L4 comprisingthe sequences of SEQ ID NOs: 48, 56, 50, and 51. In some instances, theanti-FcRH5 antibody may have a VL domain comprising the amino acidsequence of SEQ ID NO: 190. In a particular instance, the anti-FcRH5antibody can be 1G7.v1C, or a derivative or clonal relative thereof. Insome instances, for example, the invention provides an anti-FcRH5antibody having a binding domain comprising (a) a VH domain comprisingan amino acid sequence of SEQ ID NO: 189 and (b) a VL domain comprisingan amino acid sequence of SEQ ID NO: 190.

In some instances, the invention provides an anti-FcRH5 antibody havinga binding domain comprising at least one, two, three, four, five, or sixhypervariable regions (HVRs) selected from (a) an HVR-H1 comprising theamino acid sequence of SEQ ID NO: 1; (b) an HVR-H2 comprising the aminoacid sequence of SEQ ID NO: 7; (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 9; (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 11; (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 15; and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 20. In some instances, the anti-FcRH5 antibody ofclaim may have a VH domain comprising an amino acid sequence having atleast 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to, orthe sequence of, SEQ ID NO: 191 and/or a VL domain comprising an aminoacid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%)sequence identity to, or the sequence of, SEQ ID NO: 192. In someinstances, the anti-FcRH5 antibody comprises at least one (e.g., 1, 2,3, or 4) of heavy chain framework regions FR-H1, FR-H2, FR-H3, and FR-H4comprising the sequences of SEQ ID NO: 23, 54, 46, and 47. In someinstances, the anti-FcRH5 antibody may have a VH domain comprising theamino acid sequence of SEQ ID NO: 191. In some instances, the anti-FcRH5antibody further includes at least one (e.g., 1, 2, 3, or 4) of thelight chain framework regions FR-L1, FR-L2, FR-L3, and FR-L4 comprisingthe sequences of SEQ ID NOs: 48, 56, 50, and 51. In some instances, theanti-FcRH5 antibody may have a VL domain comprising the amino acidsequence of SEQ ID NO: 192. In a particular instance, the anti-FcRH5antibody can be 1G7.v1 D, or a derivative or clonal relative thereof. Insome instances, for example, the invention provides an anti-FcRH5antibody having a binding domain comprising (a) a VH domain comprisingan amino acid sequence of SEQ ID NO: 191 and (b) a VL domain comprisingan amino acid sequence of SEQ ID NO: 192.

In some instances, the invention provides an anti-FcRH5 antibody havinga binding domain comprising at least one, two, three, four, five, or sixhypervariable regions (HVRs) selected from (a) an HVR-H1 comprising theamino acid sequence of SEQ ID NO: 1; (b) an HVR-H2 comprising the aminoacid sequence of SEQ ID NO: 7; (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 9; (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 11; (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 15; and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 20. In some instances, the anti-FcRH5 antibody ofclaim may have a VH domain comprising an amino acid sequence having atleast 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to, orthe sequence of, SEQ ID NO: 193 and/or a VL domain comprising an aminoacid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%)sequence identity to, or the sequence of, SEQ ID NO: 194. In someinstances, the anti-FcRH5 antibody comprises at least one (e.g., 1, 2,3, or 4) of heavy chain framework regions FR-H1, FR-H2, FR-H3, and FR-H4comprising the sequences of SEQ ID NO: 52, 182, 46, and 47. In someinstances, the anti-FcRH5 antibody may have a VH domain comprising theamino acid sequence of SEQ ID NO: 193. In some instances, the anti-FcRH5antibody further includes at least one (e.g., 1, 2, 3, or 4) of thelight chain framework regions FR-L1, FR-L2, FR-L3, and FR-L4 comprisingthe sequences of SEQ ID NOs: 48, 56, 50, and 51. In some instances, theanti-FcRH5 antibody may have a VL domain comprising the amino acidsequence of SEQ ID NO: 194. In a particular instance, the anti-FcRH5antibody can be 1G7.v1E, or a derivative or clonal relative thereof. Insome instances, for example, the invention provides an anti-FcRH5antibody having a binding domain comprising (a) a VH domain comprisingan amino acid sequence of SEQ ID NO: 193 and (b) a VL domain comprisingan amino acid sequence of SEQ ID NO: 194.

In some instances, the invention provides an anti-FcRH5 antibody havinga binding domain comprising at least one, two, three, four, five, or sixhypervariable regions (HVRs) selected from (a) an HVR-H1 comprising theamino acid sequence of SEQ ID NO: 1; (b) an HVR-H2 comprising the aminoacid sequence of SEQ ID NO: 175; (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 9; (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 11; (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 15; and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 20. In some instances, the anti-FcRH5 antibody ofclaim may have a VH domain comprising an amino acid sequence having atleast 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to, orthe sequence of, SEQ ID NO: 195 and/or a VL domain comprising an aminoacid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%)sequence identity to, or the sequence of, SEQ ID NO: 196. In someinstances, the anti-FcRH5 antibody comprises at least one (e.g., 1, 2,3, or 4) of heavy chain framework regions FR-H1, FR-H2, FR-H3, and FR-H4comprising the sequences of SEQ ID NO: 52, 54, 46, and 47. In someinstances, the anti-FcRH5 antibody may have a VH domain comprising theamino acid sequence of SEQ ID NO: 195. In some instances, the anti-FcRH5antibody further includes at least one (e.g., 1, 2, 3, or 4) of thelight chain framework regions FR-L1, FR-L2, FR-L3, and FR-L4 comprisingthe sequences of SEQ ID NOs: 48, 56, 50, and 51. In some instances, theanti-FcRH5 antibody may have a VL domain comprising the amino acidsequence of SEQ ID NO: 196. In a particular instance, the anti-FcRH5antibody can be 1G7.v1F, or a derivative or clonal relative thereof. Insome instances, for example, the invention provides an anti-FcRH5antibody having a binding domain comprising (a) a VH domain comprisingan amino acid sequence of SEQ ID NO: 195 and (b) a VL domain comprisingan amino acid sequence of SEQ ID NO: 196.

In some instances, the invention provides an anti-FcRH5 antibody havinga binding domain comprising at least one, two, three, four, five, or sixhypervariable regions (HVRs) selected from (a) an HVR-H1 comprising theamino acid sequence of SEQ ID NO: 1; (b) an HVR-H2 comprising the aminoacid sequence of SEQ ID NO: 176; (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 9; (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 11; (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 15; and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 20. In some instances, the anti-FcRH5 antibody ofclaim may have a VH domain comprising an amino acid sequence having atleast 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to, orthe sequence of, SEQ ID NO: 197 and/or a VL domain comprising an aminoacid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%)sequence identity to, or the sequence of, SEQ ID NO: 198. In someinstances, the anti-FcRH5 antibody comprises at least one (e.g., 1, 2,3, or 4) of heavy chain framework regions FR-H1, FR-H2, FR-H3, and FR-H4comprising the sequences of SEQ ID NO: 52, 54, 46, and 47. In someinstances, the anti-FcRH5 antibody may have a VH domain comprising theamino acid sequence of SEQ ID NO: 197. In some instances, the anti-FcRH5antibody further includes at least one (e.g., 1, 2, 3, or 4) of thelight chain framework regions FR-L1, FR-L2, FR-L3, and FR-L4 comprisingthe sequences of SEQ ID NOs: 48, 56, 50, and 51. In some instances, theanti-FcRH5 antibody may have a VL domain comprising the amino acidsequence of SEQ ID NO: 198. In a particular instance, the anti-FcRH5antibody can be 1G7.v1 G, or a derivative or clonal relative thereof. Insome instances, for example, the invention provides an anti-FcRH5antibody having a binding domain comprising (a) a VH domain comprisingan amino acid sequence of SEQ ID NO: 197 and (b) a VL domain comprisingan amino acid sequence of SEQ ID NO: 198.

In some instances, the invention provides an anti-FcRH5 antibody havinga binding domain comprising at least one, two, three, four, five, or sixhypervariable regions (HVRs) selected from (a) an HVR-H1 comprising theamino acid sequence of SEQ ID NO: 1; (b) an HVR-H2 comprising the aminoacid sequence of SEQ ID NO: 177; (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 9; (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 11; (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 15; and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 20. In some instances, the anti-FcRH5 antibody ofclaim may have a VH domain comprising an amino acid sequence having atleast 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to, orthe sequence of, SEQ ID NO: 199 and/or a VL domain comprising an aminoacid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%)sequence identity to, or the sequence of, SEQ ID NO: 200. In someinstances, the anti-FcRH5 antibody comprises at least one (e.g., 1, 2,3, or 4) of heavy chain framework regions FR-H1, FR-H2, FR-H3, and FR-H4comprising the sequences of SEQ ID NO: 52, 54, 46, and 47. In someinstances, the anti-FcRH5 antibody may have a VH domain comprising theamino acid sequence of SEQ ID NO: 199. In some instances, the anti-FcRH5antibody further includes at least one (e.g., 1, 2, 3, or 4) of thelight chain framework regions FR-L1, FR-L2, FR-L3, and FR-L4 comprisingthe sequences of SEQ ID NOs: 48, 56, 50, and 51. In some instances, theanti-FcRH5 antibody may have a VL domain comprising the amino acidsequence of SEQ ID NO: 200. In a particular instance, the anti-FcRH5antibody can be 1G7.v1H, or a derivative or clonal relative thereof. Insome instances, for example, the invention provides an anti-FcRH5antibody having a binding domain comprising (a) a VH domain comprisingan amino acid sequence of SEQ ID NO: 199 and (b) a VL domain comprisingan amino acid sequence of SEQ ID NO: 200.

In some instances, the invention provides an anti-FcRH5 antibody havinga binding domain comprising at least one, two, three, four, five, or sixhypervariable regions (HVRs) selected from (a) an HVR-H1 comprising theamino acid sequence of SEQ ID NO: 1; (b) an HVR-H2 comprising the aminoacid sequence of SEQ ID NO: 178; (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 9; (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 11; (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 15; and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 20. In some instances, the anti-FcRH5 antibody ofclaim may have a VH domain comprising an amino acid sequence having atleast 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to, orthe sequence of, SEQ ID NO: 201 and/or a VL domain comprising an aminoacid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%)sequence identity to, or the sequence of, SEQ ID NO: 202. In someinstances, the anti-FcRH5 antibody comprises at least one (e.g., 1, 2,3, or 4) of heavy chain framework regions FR-H1, FR-H2, FR-H3, and FR-H4comprising the sequences of SEQ ID NO: 52, 54, 46, and 47. In someinstances, the anti-FcRH5 antibody may have a VH domain comprising theamino acid sequence of SEQ ID NO: 201. In some instances, the anti-FcRH5antibody further includes at least one (e.g., 1, 2, 3, or 4) of thelight chain framework regions FR-L1, FR-L2, FR-L3, and FR-L4 comprisingthe sequences of SEQ ID NOs: 48, 56, 50, and 51. In some instances, theanti-FcRH5 antibody may have a VL domain comprising the amino acidsequence of SEQ ID NO: 202. In a particular instance, the anti-FcRH5antibody can be 1G7.v1I, or a derivative or clonal relative thereof. Insome instances, for example, the invention provides an anti-FcRH5antibody having a binding domain comprising (a) a VH domain comprisingan amino acid sequence of SEQ ID NO: 201 and (b) a VL domain comprisingan amino acid sequence of SEQ ID NO: 202.

In some instances, the invention provides an anti-FcRH5 antibody havinga binding domain comprising at least one, two, three, four, five, or sixhypervariable regions (HVRs) selected from (a) an HVR-H1 comprising theamino acid sequence of SEQ ID NO: 1; (b) an HVR-H2 comprising the aminoacid sequence of SEQ ID NO: 7; (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 9; (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 11; (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 15; and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 20. In some instances, the anti-FcRH5 antibody ofclaim may have a VH domain comprising an amino acid sequence having atleast 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to, orthe sequence of, SEQ ID NO: 203 and/or a VL domain comprising an aminoacid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%)sequence identity to, or the sequence of, SEQ ID NO: 204. In someinstances, the anti-FcRH5 antibody comprises at least one (e.g., 1, 2,3, or 4) of heavy chain framework regions FR-H1, FR-H2, FR-H3, and FR-H4comprising the sequences of SEQ ID NO: 52, 54, 183, and 47. In someinstances, the anti-FcRH5 antibody may have a VH domain comprising theamino acid sequence of SEQ ID NO: 203. In some instances, the anti-FcRH5antibody further includes at least one (e.g., 1, 2, 3, or 4) of thelight chain framework regions FR-L1, FR-L2, FR-L3, and FR-L4 comprisingthe sequences of SEQ ID NOs: 48, 56, 50, and 51. In some instances, theanti-FcRH5 antibody may have a VL domain comprising the amino acidsequence of SEQ ID NO: 204. In a particular instance, the anti-FcRH5antibody can be 1G7.v1J, or a derivative or clonal relative thereof. Insome instances, for example, the invention provides an anti-FcRH5antibody having a binding domain comprising (a) a VH domain comprisingan amino acid sequence of SEQ ID NO: 203 and (b) a VL domain comprisingan amino acid sequence of SEQ ID NO: 204.

In some instances, the invention provides an anti-FcRH5 antibody havinga binding domain comprising at least one, two, three, four, five, or sixhypervariable regions (HVRs) selected from (a) an HVR-H1 comprising theamino acid sequence of SEQ ID NO: 1; (b) an HVR-H2 comprising the aminoacid sequence of SEQ ID NO: 7; (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 9; (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 11; (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 15; and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 20. In some instances, the anti-FcRH5 antibody ofclaim may have a VH domain comprising an amino acid sequence having atleast 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to, orthe sequence of, SEQ ID NO: 205 and/or a VL domain comprising an aminoacid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%)sequence identity to, or the sequence of, SEQ ID NO: 206. In someinstances, the anti-FcRH5 antibody comprises at least one (e.g., 1, 2,3, or 4) of heavy chain framework regions FR-H1, FR-H2, FR-H3, and FR-H4comprising the sequences of SEQ ID NO: 52, 54, 184, and 47. In someinstances, the anti-FcRH5 antibody may have a VH domain comprising theamino acid sequence of SEQ ID NO: 205. In some instances, the anti-FcRH5antibody further includes at least one (e.g., 1, 2, 3, or 4) of thelight chain framework regions FR-L1, FR-L2, FR-L3, and FR-L4 comprisingthe sequences of SEQ ID NOs: 48, 56, 50, and 51. In some instances, theanti-FcRH5 antibody may have a VL domain comprising the amino acidsequence of SEQ ID NO: 206. In a particular instance, the anti-FcRH5antibody can be 1G7.v1K, or a derivative or clonal relative thereof. Insome instances, for example, the invention provides an anti-FcRH5antibody having a binding domain comprising (a) a VH domain comprisingan amino acid sequence of SEQ ID NO: 205 and (b) a VL domain comprisingan amino acid sequence of SEQ ID NO: 206.

In some instances, the invention provides an anti-FcRH5 antibody havinga binding domain comprising at least one, two, three, four, five, or sixhypervariable regions (HVRs) selected from (a) an HVR-H1 comprising theamino acid sequence of SEQ ID NO: 1; (b) an HVR-H2 comprising the aminoacid sequence of SEQ ID NO: 7; (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 10; (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 11; (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 15; and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 20. In some instances, the anti-FcRH5 antibody ofclaim may have a VH domain comprising an amino acid sequence having atleast 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to, orthe sequence of, SEQ ID NO: 207 and/or a VL domain comprising an aminoacid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%)sequence identity to, or the sequence of, SEQ ID NO: 208. In someinstances, the anti-FcRH5 antibody comprises at least one (e.g., 1, 2,3, or 4) of heavy chain framework regions FR-H1, FR-H2, FR-H3, and FR-H4comprising the sequences of SEQ ID NO: 52, 54, 46, and 47. In someinstances, the anti-FcRH5 antibody may have a VH domain comprising theamino acid sequence of SEQ ID NO: 207. In some instances, the anti-FcRH5antibody further includes at least one (e.g., 1, 2, 3, or 4) of thelight chain framework regions FR-L1, FR-L2, FR-L3, and FR-L4 comprisingthe sequences of SEQ ID NOs: 48, 56, 50, and 51. In some instances, theanti-FcRH5 antibody may have a VL domain comprising the amino acidsequence of SEQ ID NO: 208. In a particular instance, the anti-FcRH5antibody can be 1G7.v1 L, or a derivative or clonal relative thereof. Insome instances, for example, the invention provides an anti-FcRH5antibody having a binding domain comprising (a) a VH domain comprisingan amino acid sequence of SEQ ID NO: 207 and (b) a VL domain comprisingan amino acid sequence of SEQ ID NO: 208.

In some instances, the invention provides an anti-FcRH5 antibody havinga binding domain comprising at least one, two, three, four, five, or sixhypervariable regions (HVRs) selected from (a) an HVR-H1 comprising theamino acid sequence of SEQ ID NO: 1; (b) an HVR-H2 comprising the aminoacid sequence of SEQ ID NO: 8; (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 9; (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 14; (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 16; and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 23. In some instances, the anti-FcRH5 antibody ofclaim may have a VH domain comprising an amino acid sequence having atleast 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to, orthe sequence of, SEQ ID NO: 209 and/or a VL domain comprising an aminoacid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%)sequence identity to, or the sequence of, SEQ ID NO: 210. In someinstances, the anti-FcRH5 antibody comprises at least one (e.g., 1, 2,3, or 4) of heavy chain framework regions FR-H1, FR-H2, FR-H3, and FR-H4comprising the sequences of SEQ ID NO: 52, 54, 46, and 47. In someinstances, the anti-FcRH5 antibody may have a VH domain comprising theamino acid sequence of SEQ ID NO: 209. In some instances, the anti-FcRH5antibody further includes at least one (e.g., 1, 2, 3, or 4) of thelight chain framework regions FR-L1, FR-L2, FR-L3, and FR-L4 comprisingthe sequences of SEQ ID NOs: 48, 56, 50, and 51. In some instances, theanti-FcRH5 antibody may have a VL domain comprising the amino acidsequence of SEQ ID NO: 210. In a particular instance, the anti-FcRH5antibody can be 1G7.v86, or a derivative or clonal relative thereof. Insome instances, for example, the invention provides an anti-FcRH5antibody having a binding domain comprising (a) a VH domain comprisingan amino acid sequence of SEQ ID NO: 209 and (b) a VL domain comprisingan amino acid sequence of SEQ ID NO: 210.

In some instances, the invention provides an anti-FcRH5 antibody havinga binding domain comprising at least one, two, three, four, five, or sixhypervariable regions (HVRs) selected from (a) an HVR-H1 comprising theamino acid sequence of SEQ ID NO: 1; (b) an HVR-H2 comprising the aminoacid sequence of SEQ ID NO: 8; (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 9; (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 14; (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 16; and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 23. In some instances, the anti-FcRH5 antibody ofclaim may have a VH domain comprising an amino acid sequence having atleast 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to, orthe sequence of, SEQ ID NO: 211 and/or a VL domain comprising an aminoacid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%)sequence identity to, or the sequence of, SEQ ID NO: 212. In someinstances, the anti-FcRH5 antibody comprises at least one (e.g., 1, 2,3, or 4) of heavy chain framework regions FR-H1, FR-H2, FR-H3, and FR-H4comprising the sequences of SEQ ID NO: 53, 54, 46, and 47. In someinstances, the anti-FcRH5 antibody may have a VH domain comprising theamino acid sequence of SEQ ID NO: 211. In some instances, the anti-FcRH5antibody further includes at least one (e.g., 1, 2, 3, or 4) of thelight chain framework regions FR-L1, FR-L2, FR-L3, and FR-L4 comprisingthe sequences of SEQ ID NOs: 48, 56, 50, and 51. In some instances, theanti-FcRH5 antibody may have a VL domain comprising the amino acidsequence of SEQ ID NO: 212. In a particular instance, the anti-FcRH5antibody can be 1G7.v191, or a derivative or clonal relative thereof. Insome instances, for example, the invention provides an anti-FcRH5antibody having a binding domain comprising (a) a VH domain comprisingan amino acid sequence of SEQ ID NO: 211 and (b) a VL domain comprisingan amino acid sequence of SEQ ID NO: 212.

In some instances, the invention provides an anti-FcRH5 antibody havinga binding domain comprising at least one, two, three, four, five, or sixhypervariable regions (HVRs) selected from (a) an HVR-H1 comprising theamino acid sequence of SEQ ID NO: 1; (b) an HVR-H2 comprising the aminoacid sequence of SEQ ID NO: 8; (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 10; (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 14; (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 16; and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 23. In some instances, the anti-FcRH5 antibody ofclaim may have a VH domain comprising an amino acid sequence having atleast 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to, orthe sequence of, SEQ ID NO: 213 and/or a VL domain comprising an aminoacid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%)sequence identity to, or the sequence of, SEQ ID NO: 214. In someinstances, the anti-FcRH5 antibody comprises at least one (e.g., 1, 2,3, or 4) of heavy chain framework regions FR-H1, FR-H2, FR-H3, and FR-H4comprising the sequences of SEQ ID NO: 52, 54, 46, and 47. In someinstances, the anti-FcRH5 antibody may have a VH domain comprising theamino acid sequence of SEQ ID NO: 213. In some instances, the anti-FcRH5antibody further includes at least one (e.g., 1, 2, 3, or 4) of thelight chain framework regions FR-L1, FR-L2, FR-L3, and FR-L4 comprisingthe sequences of SEQ ID NOs: 48, 56, 50, and 51. In some instances, theanti-FcRH5 antibody may have a VL domain comprising the amino acidsequence of SEQ ID NO: 214. In a particular instance, the anti-FcRH5antibody can be 1G7.v92, or a derivative or clonal relative thereof. Insome instances, for example, the invention provides an anti-FcRH5antibody having a binding domain comprising (a) a VH domain comprisingan amino acid sequence of SEQ ID NO: 213 and (b) a VL domain comprisingan amino acid sequence of SEQ ID NO: 214.

In certain embodiments, an antibody provided herein is a monoclonal,human, humanized, or chimeric antibody. In some instances, theanti-FcRH5 antibody is an IgG antibody. The anti-FcRH5 may be afull-length antibody and/or a monospecific antibody. In certainembodiments, the anti-FcRH5 antibody may bind to an epitope in theIg-like domain 9 of FcRH5. For example, the epitope may comprises aportion of amino acids 743-850 of SEQ ID NO: 114. In some instances, theanti-FcRH5 antibody binds to human FcRH5 or cynomolgus monkey (cyno)FcRH5, or both. In other instances, the binding domain does notspecifically bind to FcRH1, FcRH2, FcRH3, and/or FcRH4.

In some instances, the anti-FcRH5 antibody has a clearance followingintravenous injection of between about 10 ml/kg/day to about 45ml/kg/day (e.g., about 1 ml/kg/day, 5 ml/kg/day, 10 ml/kg/day, 11ml/kg/day, 12 ml/kg/day, 13 ml/kg/day, 14 ml/kg/day, 15 ml/kg/day, 16ml/kg/day, 17 ml/kg/day, 18 ml/kg/day, 19 ml/kg/day, 20 ml/kg/day, 21ml/kg/day, 22 ml/kg/day, 23 ml/kg/day, 24 ml/kg/day, 25 ml/kg/day, 26ml/kg/day, 27 ml/kg/day, 28 ml/kg/day, 29 ml/kg/day, 30 ml/kg/day, 31ml/kg/day, 32 ml/kg/day, 33 ml/kg/day, 34 ml/kg/day, 35 ml/kg/day, 36ml/kg/day, 37 ml/kg/day, 38 ml/kg/day, 39 ml/kg/day, 40 ml/kg/day, 41ml/kg/day, 42 ml/kg/day, 43 ml/kg/day, or 44 ml/kg/day).

In some instances, the anti-FcRH5 antibody has a clearance followingintravenous injection of about 1 ml/kg/day to about 5 ml/kg/day, about 6ml/kg/day to about 10 ml/kg/day, about 11 ml/kg/day to about 15ml/kg/day, about 16 ml/kg/day to about 20 ml/kg/day, about 21 ml/kg/dayto about 25 ml/kg/day, about 26 ml/kg/day to about 30 ml/kg/day, about31 ml/kg/day to about 35 ml/kg/day, about 36 ml/kg/day to about 40ml/kg/day, about 41 ml/kg/day to about 45 ml/kg/day in a mouse. In someinstances, the anti-FcRH5 antibody has a clearance following intravenousinjection of about 10 ml/kg/day to about 35 ml/kg/day in a mouse. Insome instances, the anti-FcRH5 antibody has a clearance followingintravenous injection of about 10 ml/kg/day to about 20 ml/kg/day in amouse. In some instances, the anti-FcRH5 antibody has a clearancefollowing intravenous injection of about 12 ml/kg/day to about 16ml/kg/day in a mouse.

In some instances, the anti-FcRH5 antibody has a clearance followingintravenous injection of about 1 ml/kg/day to about 5 ml/kg/day, about 6ml/kg/day to about 10 ml/kg/day, about 11 ml/kg/day to about 15ml/kg/day, about 16 ml/kg/day to about 20 ml/kg/day, about 21 ml/kg/dayto about 25 ml/kg/day, about 26 ml/kg/day to about 30 ml/kg/day, about31 ml/kg/day to about 35 ml/kg/day, about 36 ml/kg/day to about 40ml/kg/day, about 41 ml/kg/day to about 45 ml/kg/day in cyno. In someinstances, the anti-FcRH5 antibody has a clearance following intravenousinjection of about 20 ml/kg/day to about 40 ml/kg/day in a cyno. In someinstances, the anti-FcRH5 antibody has a clearance following intravenousinjection of about 25 ml/kg/day to about 35 ml/kg/day in a cyno. In someinstances, the anti-FcRH5 antibody has a clearance following intravenousinjection of about 30 ml/kg/day to about 35 ml/kg/day in a cyno.

In a further aspect, an anti-FcRH5 antibody according to any of theabove embodiments may incorporate any of the features, singly or incombination, as described in Sections 1-7 below.

1. Antibody Affinity

In particular instances, the anti-FcRH5 antibody binds to human and/orcyno FcRH5 with a dissociation constant (K_(D)) of ≤1 μM, ≤100 nM, ≤10nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10⁻⁸ M or less, e.g.,from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ M to 10⁻¹³ M, e.g., from 10⁻¹⁰to 10⁻¹³ M, e.g., from 10⁻¹¹ to 10⁻¹³ M, e.g., from 10⁻¹² to 10⁻¹³ M).For example, in some instances the anti-FcRH5 antibody binds to humanFcRH5 with a K_(D) of ≤100 nM (e.g, ≤90 nM, ≤80 nM, ≤70 nM, ≤60 nM, ≤50nM, ≤40 nM, 530 nM, ≤20 nM, ≤10 nM, ≤5 nM, ≤1 nM, ≤750 pM, ≤500 pM, ≤250pM, ≤100 pM, ≤50 pM, 525 pM, ≤10 pM, 55 pM, or ≤1 pM) or lower.

In some instances, the anti-FcRH5 antibody binds to human FcRH5 with aK_(D) of about 1 pM to about 500 nM (e.g., about 1 pM to 200 pM, 100 pMto 300 pM, 200 pM to 400 pM, 300 pM to 500 pM, 400 pM to 600 pM, 500 pMto 700 pM, 600 pM to 800 pM, 700 pM to 900 pM, 800 pM to 1 nM, 900 pM to100 nM, 1 nM to 200 nM, 100 nM to 300 nM, 200 nM to 400 nM, or 300 nM to500 nM). In some instances, the anti-FcRH5 antibody binds to human FcRH5with a K_(D) of about 1 pM to about 1 nM (e.g., about 1 pM to 100 pM, 50pM to 150 pM, 100 pM to 200 pM, 150 pM to 250 pM, 200 pM to 300 pM, 250pM to 350 pM, 300 pM to 400 pM, 350 pM to 450 pM, 400 pM to 500 pM, 450pM to 550 pM, 500 pM to 600 pM, 550 pM to 650 pM, 600 pM to 700 pM, 650pM to 750 pM, 700 pM to 800 pM, 750 pM to 850 pM, 800 pM to 900 pM, 850pM to 950 pM, or 900 pM to 1 nM). In some instances, the anti-FcRH5antibody binds to human FcRH5 with a K_(D) of about 100 pM to about 500pM (e.g., about 100 pM, 125 pM, 150 pM, 175 pM, 200 pM, 225 pM, 250 pM,275 pM, 300 pM, 325 pM, 350 pM, 375 pM, 400 pM, 425 pM, 450 pM, 475 pM,or 500 pM). In some instances, the anti-FcRH5 antibody binds to humanFcRH5 with a K_(D) of about 100 pM to about 160 pM (e.g., about 100 pM,105 pM, 110 pM, 115 pM, 120 pM, 125 pM, 130 pM, 135 pM, 140 pM, 145 pM,150 pM, 155 pM, or 160 pM). In some instances, the anti-FcRH5 antibodybinds to human FcRH5 with a K_(D) of about 1 nM to about 150 nM (e.g.,about 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 11nM, 12, nM, 13 nM, 14 nM, 15 nM, 16 nM, 17 nM, 18 nM, 19 nM, 20 nM, 21nM, 22 nM, 23 nM, 24 nM, 25 nM, 26 nM, 27 nM, 28 nM, 29 nM, 30 nM, 31nM, 32 nM, 33 nM, 34 nM, 35 nM, 36 nM, 37 nM, 38 nM, 39 nM, 40 nM, 41nM, 42 nM, 43 nM, 44 nM, 45 nM, 46 nM, 47 nM, 48 nM, 49 nM, 50 nM, 55nM, 60 nM, 65 nM, 70 nM, 75 nM, 80 nM, 85 nM, 90 nM, 95 nM, 100 nM, 105nM, 110 nM, 115 nM, 120 nM, 125 nM, 130 nM, 135 nM, 140 nM, 145 nM, or150 nM).

In some instances the anti-FcRH5 antibody binds to cyno FcRH5 with aK_(D) of ≤100 nM (e.g, ≤90 nM, ≤80 nM, ≤70 nM, ≤60 nM, ≤50 nM, ≤40 nM,≤30 nM, ≤20 nM, ≤10 nM, ≤5 nM, ≤1 nM, ≤750 pM, ≤500 pM, ≤250 pM, ≤100pM, ≤50 pM, ≤25 pM, ≤10 pM, ≤5 pM, or ≤1 pM, e.g., from 10⁻⁸M to 10⁻¹³M, e.g., from 10⁻⁹ M to 10⁻¹³ M, e.g., from 10⁻¹⁰ to 10⁻¹³ M, e.g., from10⁻¹¹ to 10⁻¹³M, e.g., from 10⁻¹² to 10⁻¹³M) or lower. In someinstances, the anti-FcRH5 antibody binds to cyno FcRH5 with a K_(D) ofabout 1 pM to about 500 nM (e.g., about 1 pM to 200 pM, 100 pM to 300pM, 200 pM to 400 pM, 300 pM to 500 pM, 400 pM to 600 pM, 500 pM to 700pM, 600 pM to 800 pM, 700 pM to 900 pM, 800 pM to 1 nM, 900 pM to 100nM, 1 nM to 200 nM, 100 nM to 300 nM, 200 nM to 400 nM, or 300 nM to 500nM). In some instances, the anti-FcRH5 antibody binds to cyno FcRH5 witha K_(D) of about 1 pM to about 1 nM (e.g., about 1 pM to 100 pM, 50 pMto 150 pM, 100 pM to 200 pM, 150 pM to 250 pM, 200 pM to 300 pM, 250 pMto 350 pM, 300 pM to 400 pM, 350 pM to 450 pM, 400 pM to 500 pM, 450 pMto 550 pM, 500 pM to 600 pM, 550 pM to 650 pM, 600 pM to 700 pM, 650 pMto 750 pM, 700 pM to 800 pM, 750 pM to 850 pM, 800 pM to 900 pM, 850 pMto 950 pM, or 900 pM to 1 nM). In some instances, the anti-FcRH5antibody binds to cyno FcRH5 with a K_(D) of about 100 pM to about 500pM (e.g., about 100 pM, 125 pM, 150 pM, 175 pM, 200 pM, 225 pM, 250 pM,275 pM, 300 pM, 325 pM, 350 pM, 375 pM, 400 pM, 425 pM, 450 pM, 475 pM,or 500 pM). In some instances, the anti-FcRH5 antibody binds to cynoFcRH5 with a K_(D) of about 100 pM to about 160 pM (e.g., about 100 pM,105 pM, 110 pM, 115 pM, 120 pM, 125 pM, 130 pM, 135 pM, 140 pM, 145 pM,150 pM, 155 pM, or 160 pM). In some instances, the anti-FcRH5 antibodybinds to cyno FcRH5 with a K_(D) of about 1 nM to about 150 nM (e.g.,about 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 11nM, 12, nM, 13 nM, 14 nM, 15 nM, 16 nM, 17 nM, 18 nM, 19 nM, 20 nM, 21nM, 22 nM, 23 nM, 24 nM, 25 nM, 26 nM, 27 nM, 28 nM, 29 nM, 30 nM, 31nM, 32 nM, 33 nM, 34 nM, 35 nM, 36 nM, 37 nM, 38 nM, 39 nM, 40 nM, 41nM, 42 nM, 43 nM, 44 nM, 45 nM, 46 nM, 47 nM, 48 nM, 49 nM, 50 nM, 55nM, 60 nM, 65 nM, 70 nM, 75 nM, 80 nM, 85 nM, 90 nM, 95 nM, 100 nM, 105nM, 110 nM, 115 nM, 120 nM, 125 nM, 130 nM, 135 nM, 140 nM, 145 nM, or150 nM).

In one embodiment, K_(D) is measured by a radiolabeled antigen bindingassay (RIA). In one embodiment, an RIA is performed with the Fab versionof an antibody of interest and its antigen. For example, solutionbinding affinity of Fabs for antigen is measured by equilibrating Fabwith a minimal concentration of (¹²⁵I)-labeled antigen in the presenceof a titration series of unlabeled antigen, then capturing bound antigenwith an anti-Fab antibody-coated plate (see, e.g., Chen. et al. J. Mol.Biol. 293:865-881, 1999). To establish conditions for the assay,MICROTITER® multi-well plates (Thermo Scientific) are coated overnightwith 5 μg/ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mMsodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovineserum albumin in PBS for two to five hours at room temperature(approximately 23° C.). In a non-adsorbent plate (Nunc #269620), 100 pMor 26 pM [¹²⁵1]-antigen are mixed with serial dilutions of a Fab ofinterest (e.g., consistent with assessment of the anti-VEGF antibody,Fab-12, in Presta et al. Cancer Res. 57:4593-4599, 1997). The Fab ofinterest is then incubated overnight; however, the incubation maycontinue for a longer period (e.g., about 65 hours) to ensure thatequilibrium is reached. Thereafter, the mixtures are transferred to thecapture plate for incubation at room temperature (e.g., for one hour).The solution is then removed and the plate washed eight times with 0.1%polysorbate 20 (TWEEN-20®) in PBS. When the plates have dried, 150μl/well of scintillant (MICROSCINT-20™; Packard) is added, and theplates are counted on a TOPCOUNT™ gamma counter (Packard) for tenminutes. Concentrations of each Fab that give less than or equal to 20%of maximal binding are chosen for use in competitive binding assays.

According to another embodiment, K_(D) is measured using a BIACORE®surface plasmon resonance assay. For example, an assay using aBIACORE®-2000 or a BIACORE®-3000 (BIAcore, Inc., Piscataway, N.J.) isperformed at 25° C. with immobilized antigen CM5 chips at 10 responseunits (RU). In one embodiment, carboxymethylated dextran biosensor chips(CM5, BIACORE, Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (˜0.2μM) before injection at a flow rate of 5 μl/minute to achieveapproximately 10 response units (RU) of coupled protein. Following theinjection of antigen, 1 M ethanolamine is injected to block unreactedgroups. For kinetics measurements, two-fold serial dilutions of Fab(0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20(TWEEN-20™) surfactant (PBST) at 25° C. at a flow rate of approximately25 μl/min. Association rates (k_(on)) and dissociation rates (k_(on))are calculated using a simple one-to-one Langmuir binding model(BIACORE® Evaluation Software version 3.2) by simultaneously fitting theassociation and dissociation sensorgrams. The equilibrium dissociationconstant (K_(D)) is calculated as the ratio k_(off)/k_(on). See, forexample, Chen et al. J. Mol. Biol. 293:865-881, 1999. If the on-rateexceeds 10⁶M⁻¹ s⁻¹ by the surface plasmon resonance assay above, thenthe on-rate can be determined by using a fluorescent quenching techniquethat measures the increase or decrease in fluorescence emissionintensity (excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25°C. of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in thepresence of increasing concentrations of antigen as measured in aspectrometer, such as a stop-flow equipped spectrophometer (AvivInstruments) or a 8000-series SLM-AMINCO™ spectrophotometer(ThermoSpectronic) with a stirred cuvette.

2. Antibody Fragments

In certain embodiments, an antibody provided herein is an antibodyfragment. Antibody fragments include, but are not limited to, bis-Fabs,Fab, Fab′, Fab′-SH, F(ab′)₂, Fv, and scFv fragments, and other fragmentsdescribed below. For a review of certain antibody fragments, see Hudsonet al. Nat. Med. 9:129-134, 2003. For a review of scFv fragments, see,e.g., Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315,1994; see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458.For discussion of Fab and F(ab′)₂ fragments comprising salvage receptorbinding epitope residues and having increased in vivo half-life, seeU.S. Pat. No. 5,869,046.

Antibody fragments in which the two Fabs are linked throughbis-maleimide are referred to herein as bismaleimido-(thio-Fab)₂ orbis-Fabs.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al. Nat. Med. 9:129-134, 2003; and Hollinger et al. Proc.Natl. Acad. Sci. USA 90:6444-6448, 1993. Triabodies and tetrabodies arealso described in Hudson et al. Nat. Med. 9:129-134, 2003.

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g., E. coli or phage), asdescribed herein.

3. Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimericantibody. Certain chimeric antibodies are described, e.g., in U.S. Pat.No. 4,816,567; and Morrison et al. Proc. Natl. Acad. Sci. USA,81:6851-6855, 1984. In one example, a chimeric antibody comprises anon-human variable region (e.g., a variable region derived from a mouse,rat, hamster, rabbit, or non-human primate, such as a monkey) and ahuman constant region. In a further example, a chimeric antibody is a“class switched” antibody in which the class or subclass has beenchanged from that of the parent antibody. Chimeric antibodies includeantigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, e.g., CDRs, (or portions thereof)are derived from a non-human antibody, and FRs (or portions thereof) arederived from human antibody sequences. A humanized antibody optionallywill also comprise at least a portion of a human constant region. Insome embodiments, some FR residues in a humanized antibody aresubstituted with corresponding residues from a non-human antibody (e.g.,the antibody from which the HVR residues are derived), e.g., to restoreor improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro et al. Front. Biosci. 13:1619-1633, 2008, and are furtherdescribed, e.g., in Riechmann et al. Nature 332:323-329, 1988; Queen etal. Proc. Natl Acad. Sci. USA 86:10029-10033, 1989; US Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al. Methods36:25-34, 2005 (describing specificity determining region (SDR)grafting); Padlan Mol. Immunol. 28:489-498, 1991 (describing“resurfacing”); Dall′Acqua et al. Methods 36:43-60, 2005 (describing “FRshuffling”); and Osbourn et al. Methods 36:61-68, 2005; and Klimka etal. Br. J. Cancer, 83:252-260, 2000 (describing the “guided selection”approach to FR shuffling).

Human framework regions that may be used for humanization include, butare not limited to, framework regions selected using the “best-fit”method (see, e.g., Sims et al. J. Immunol. 151:2296, 1993); frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light or heavy chain variable regions (see, e.g.,Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285, 1992; and Presta etal. J. Immunol. 151:2623, 1993); human mature (somatically mutated)framework regions or human germline framework regions (see, e.g.,Almagro et al. Front. Biosci. 13:1619-1633, 2008); and framework regionsderived from screening FR libraries (see, e.g., Baca et al. J. Biol.Chem. 272:10678-10684, 1997 and Rosok et al. J. Biol. Chem.271:22611-22618, 1996).

4. Human Antibodies

In certain embodiments, an antibody provided herein is a human antibody.Human antibodies can be produced using various techniques known in theart. Human antibodies are described generally in van Dijk and van deWinkel, Curr. Opin. Pharmacol. 5: 368-74, 2001 and Lonberg, Curr. Opin.Immunol. 20:450-459, 2008.

Human antibodies may be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125, 2005. Seealso, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™technology; U.S. Pat. No. 5,770,429 describing HUMAB® technology; U.S.Pat. No. 7,041,870 describing K-M MOUSE® technology, and U.S. PatentApplication Publication No. US 2007/0061900, describing VELOCIMOUSE®technology). Human variable regions from intact antibodies generated bysuch animals may be further modified, e.g., by combining with adifferent human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described (see, e.g., Kozbor J.Immunol., 133: 3001, 1984; Brodeur et al. Monoclonal Antibody ProductionTechniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York,1987); and Boerner et al. J. Immunol., 147: 86, 1991). Human antibodiesgenerated via human B-cell hybridoma technology are also described in Liet al. Proc. Natl. Acad. Sci. USA, 103:3557-3562, 2006. Additionalmethods include those described, for example, in U.S. Pat. No. 7,189,826(describing production of monoclonal human IgM antibodies from hybridomacell lines) and Ni, Xiandai Mianyixue, 26(4):265-268, 2006 (describinghuman-human hybridomas). Human hybridoma technology (Trioma technology)is also described in Vollmers and Brandlein, Histology andHistopathology, 20(3):927-937, 2005 and Vollmers and Brandlein, Methodsand Findings in Experimental and Clinical Pharmacology, 27(3):185-91,2005.

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described below.

5. Library-Derived Antibodies

Antibodies of the invention may be isolated by screening combinatoriallibraries for antibodies with the desired activity or activities. Forexample, a variety of methods are known in the art for generating phagedisplay libraries and screening such libraries for antibodies possessingthe desired binding characteristics. Such methods are reviewed, e.g., inHoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien etal. ed., Human Press, Totowa, N.J., 2001) and further described, e.g.,in the McCafferty et al. Nature 348:552-554; Clackson et al. Nature 352:624-628, 1991; Marks et al. J. Mol. Biol. 222: 581-597, 1992; Marks andBradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., HumanPress, Totowa, N.J., 2003); Sidhu et al. J. Mol. Biol. 338(2): 299-310,2004; Lee et al. J. Mol. Biol. 340(5):1073-1093, 2004; Fellouse, Proc.Natl. Acad. Sci. USA 101(34): 12467-12472, 2004; and Lee et al. J.Immunol. Methods 284(1-2):119-132, 2004.

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al. Ann. Rev. Immunol.,12: 433-455, 1994. Phage typically display antibody fragments, either assingle-chain Fv (scFv) fragments or as Fab fragments. Libraries fromimmunized sources provide high-affinity antibodies to the immunogenwithout the requirement of constructing hybridomas. Alternatively, thenaive repertoire can be cloned (e.g., from human) to provide a singlesource of antibodies to a wide range of non-self and also self antigenswithout any immunization as described by Griffiths et al. EMBO J, 12:725-734, 1993. Finally, naive libraries can also be made syntheticallyby cloning unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro, as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388, 1992. Patentpublications describing human antibody phage libraries include, forexample: U.S. Pat. No. 5,750,373, and US Patent Publication Nos.2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,2007/0237764, 2007/0292936, and 2009/0002360. Antibodies or antibodyfragments isolated from human antibody libraries are considered humanantibodies or human antibody fragments herein.

6. Multispecific Antibodies, Including FcRH5 T Cell-Dependent Bispecific(TDB) Antibodies

In any one of the above aspects, the anti-FcRH5 antibody provided hereinis a multispecific antibody, for example, a bispecific antibody.Multispecific antibodies are monoclonal antibodies that have bindingspecificities for at least two different sites. In certain embodiments,bispecific antibodies may bind to two different epitopes of FcRH5.

In certain embodiments, one of the binding specificities is for FcRH5and the other is for CD3 (e.g., CD3ε or CD3γ). Such bispecificanti-FcRH5 antibodies are also referred to as FcRH5 T cell-dependentbispecific (TDB) antibodies or FcRH5 TDBs. In some instances, the secondbinding domain binds to an epitope on CD3 comprising amino acid residueGlu6 of CD3. In some instances, the epitope further comprises one ormore additional amino acid residues selected from the group consistingof Gln1, Asp2, and Met7 of CD3. In some instances, the epitope comprisesamino acid residues Gln1, Asp2, and Glu6 of CD3. In some instances, theepitope comprises amino acid residues Gln1, Asp2, Glu6, and Met7 of CD3.In some instances, the epitope does not comprise amino acid residue Glu5of CD3. In some instances, the epitope does not comprise amino acidresidues Gly3 and Glu5 of CD3. In some instances, the epitope consistsof amino acid residues Gln1, Asp2, Glu6, and Met7 of CD3.

In other some instances, the second binding domain is capable of bindingto a human CD3 polypeptide or a cyno CD3 polypeptide. In some instances,the human CD3 polypeptide or the cyno CD3 polypeptide is a human CD3εpolypeptide or a cyno CD3ε polypeptide, respectively. In some instances,the human CD3 polypeptide or the cyno CD3 polypeptide is a human CD3γpolypeptide or a cyno CD3γ polypeptide, respectively.

In particular instances, the second binding domain binds the human CD3εpolypeptide with dissociation constant (K_(D)) of ≤1 μM, ≤100 nM, ≤10nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10⁻⁸ M or less, e.g.,from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ M to 10⁻¹³ M). For example, insome instances the the second binding domain binds the human CD3εpolypeptide with a K_(D) of ≤100 nM (e.g, ≤90 nM, 580 nM, ≤70 nM, ≤60nM, ≤50 nM, ≤40 nM, ≤30 nM, ≤20 nM, ≤10 nM, ≤5 nM, ≤1 nM, ≤750 μM, ≤500μM, ≤250 μM, ≤100 μM, ≤50 μM, ≤25 μM, ≤10 μM, ≤5 μM, or ≤1 μM) or lower.

In some instances, for example, the invention provides an anti-FcRH5antibody, wherein the second binding domain comprises at least one, two,three, four, five, or six hypervariable regions (HVRs) selected from (a)an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 115; (b) anHVR-H2 comprising the amino acid sequence of SEQ ID NO: 116; (c) anHVR-H3 comprising the amino acid sequence of SEQ ID NO: 117; (d) anHVR-L1 comprising the amino acid sequence of SEQ ID NO: 118; (e) anHVR-L2 comprising the amino acid sequence of SEQ ID NO: 119; and (f) anHVR-L3 comprising the amino acid sequence of SEQ ID NO: 120.

In some instances, the invention provides an anti-FcRH5 antibody,wherein the second binding domain comprises at least one, two, three,four, five, or six hypervariable regions (HVRs) selected from (a) anHVR-H1 comprising the amino acid sequence of SEQ ID NO: 115; (b) anHVR-H2 comprising the amino acid sequence of SEQ ID NO: 116; (c) anHVR-H3 comprising the amino acid sequence of SEQ ID NO: 121; (d) anHVR-L1 comprising the amino acid sequence of SEQ ID NO: 118; (e) anHVR-L2 comprising the amino acid sequence of SEQ ID NO: 119; and (f) anHVR-L3 comprising the amino acid sequence of SEQ ID NO: 123. In someinstances, the invention provides an anti-FcRH5 antibody, wherein thesecond binding domain includes a VH domain comprising an amino acidsequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%)sequence identity to, or the sequence of, SEQ ID NO: 133 and/or a VLdomain comprising an amino acid sequence having at least 80% (e.g., 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99%) sequence identity to, or the sequence of,SEQ ID NO: 134. In some instances, the anti-FcRH5 antibody includes asecond binding domain comprising at least one (e.g., 1, 2, 3, or 4) ofheavy chain framework regions FR-H1, FR-H2, FR-H3, and FR-H4 comprisingthe sequences of SEQ ID NOs: 125, 126, 127, and 128, respectively. Insome instances, the second binding domain comprises a VH domaincomprising the amino acid sequence of SEQ ID NO: 133. In some instances,the second binding domain further includes at least one (e.g., 1, 2, 3,or 4) of the light chain framework regions FR-L1, FR-L2, FR-L3, andFR-L4 comprising the sequences of SEQ ID NOs: 129, 130, 131, and 132,respectively. In some instances, the second binding domain comprises aVL domain comprising the amino acid sequence of SEQ ID NO: 134. In someinstances, the second binding domain includes a VH domain comprising anamino acid sequence of SEQ ID NO: 133 and (b) a VL domain comprising anamino acid sequence of SEQ ID NO: 134. Accordingly, in some instances, ahalf-antibody variant of an anti-FcRH5 antibody of the invention may bepaired with a half-antibody variant of anti-CD3 antibody 38E4.v1 to forman FcRH5 TDB (i.e., an anti-FcRH5/38E4.v1 TDB).

In some instances, the invention provides an anti-FcRH5 antibody,wherein the second binding domain comprises at least one, two, three,four, five, or six hypervariable regions (HVRs) selected from (a) anHVR-H1 comprising the amino acid sequence of SEQ ID NO: 115; (b) anHVR-H2 comprising the amino acid sequence of SEQ ID NO: 116; (c) anHVR-H3 comprising the amino acid sequence of SEQ ID NO: 122; (d) anHVR-L1 comprising the amino acid sequence of SEQ ID NO: 118; (e) anHVR-L2 comprising the amino acid sequence of SEQ ID NO: 119; and (f) anHVR-L3 comprising the amino acid sequence of SEQ ID NO: 123. In someinstances, the invention provides an anti-FcRH5 antibody, wherein thesecond binding domain includes a VH domain comprising an amino acidsequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%)sequence identity to, or the sequence of, SEQ ID NO: 135 and/or a VLdomain comprising an amino acid sequence having at least 80% (e.g., 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99%) sequence identity to, or the sequence of,SEQ ID NO: 136. In some instances, the anti-FcRH5 antibody includes asecond binding domain comprising at least one (e.g., 1, 2, 3, or 4) ofheavy chain framework regions FR-H1, FR-H2, FR-H3, and FR-H4 comprisingthe sequences of SEQ ID NOs: 125, 126, 127, and 128, respectively. Insome instances, the second binding domain comprises a VH domaincomprising the amino acid sequence of SEQ ID NO: 135. In some instances,the second binding domain further includes at least one (e.g., 1, 2, 3,or 4) of the light chain framework regions FR-L1, FR-L2, FR-L3, andFR-L4 comprising the sequences of SEQ ID NOs: 129, 130, 131, and 132,respectively. In some instances, the second binding domain comprises aVL domain comprising the amino acid sequence of SEQ ID NO: 136. In someinstances, the second binding domain includes a VH domain comprising anamino acid sequence of SEQ ID NO: 135 and (b) a VL domain comprising anamino acid sequence of SEQ ID NO: 136. Accordingly, in some instances, ahalf-antibody variant of an anti-FcRH5 antibody of the invention may bepaired with a half-antibody variant of anti-CD3 antibody 38E4.v1 to forman FcRH5 TDB (i.e., an anti-FcRH5/38E4.v1 TDB).

In some instances, the invention provides an anti-FcRH5 antibody,wherein the second binding domain comprises at least one, two, three,four, five, or six hypervariable regions (HVRs) selected from (a) anHVR-H1 comprising the amino acid sequence of SEQ ID NO: 115; (b) anHVR-H2 comprising the amino acid sequence of SEQ ID NO: 116; (c) anHVR-H3 comprising the amino acid sequence of SEQ ID NO: 121; (d) anHVR-L1 comprising the amino acid sequence of SEQ ID NO: 118; (e) anHVR-L2 comprising the amino acid sequence of SEQ ID NO: 119; and (f) anHVR-L3 comprising the amino acid sequence of SEQ ID NO: 124. In someinstances, the invention provides an anti-FcRH5 antibody, wherein thesecond binding domain includes a VH domain comprising an amino acidsequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%)sequence identity to, or the sequence of, SEQ ID NO: 137 and/or a VLdomain comprising an amino acid sequence having at least 80% (e.g., 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99%) sequence identity to, or the sequence of,SEQ ID NO: 138. In some instances, the anti-FcRH5 antibody includes asecond binding domain comprising at least one (e.g., 1, 2, 3, or 4) ofheavy chain framework regions FR-H1, FR-H2, FR-H3, and FR-H4 comprisingthe sequences of SEQ ID NOs: 125, 126, 127, and 128, respectively. Insome instances, the second binding domain comprises a VH domaincomprising the amino acid sequence of SEQ ID NO: 137. In some instances,the second binding domain further includes at least one (e.g., 1, 2, 3,or 4) of the light chain framework regions FR-L1, FR-L2, FR-L3, andFR-L4 comprising the sequences of SEQ ID NOs: 129, 130, 131, and 132,respectively. In some instances, the second binding domain comprises aVL domain comprising the amino acid sequence of SEQ ID NO: 138. In someinstances, the second binding domain includes a VH domain comprising anamino acid sequence of SEQ ID NOs: 137 and (b) a VL domain comprising anamino acid sequence of SEQ ID NO: 138. Accordingly, in some instances, ahalf-antibody variant of an anti-FcRH5 antibody of the invention may bepaired with a half-antibody variant of anti-CD3 antibody 38E4.v11 toform an FcRH5 TDB (i.e., an anti-FcRH5/38E4.v11 TDB).

In some instances, the invention provides an anti-FcRH5 antibody,wherein the second binding domain comprises at least one, two, three,four, five, or six hypervariable regions (HVRs) selected from (a) anHVR-H1 comprising the amino acid sequence of SEQ ID NO: 139; (b) anHVR-H2 comprising the amino acid sequence of SEQ ID NO: 140; (c) anHVR-H3 comprising the amino acid sequence of SEQ ID NO: 141; (d) anHVR-L1 comprising the amino acid sequence of SEQ ID NO: 142; (e) anHVR-L2 comprising the amino acid sequence of SEQ ID NO: 143; and (f) anHVR-L3 comprising the amino acid sequence of SEQ ID NO: 144. In someinstances, the invention provides an anti-FcRH5 antibody, wherein thesecond binding domain includes a VH domain comprising an amino acidsequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%)sequence identity to, or the sequence of, SEQ ID NO: 153 and/or a VLdomain comprising an amino acid sequence having at least 80% (e.g., 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99%) sequence identity to, or the sequence of,SEQ ID NO: 154. In some instances, the anti-FcRH5 antibody includes asecond binding domain comprising at least one (e.g., 1, 2, 3, or 4) ofheavy chain framework regions FR-H1, FR-H2, FR-H3, and FR-H4 comprisingthe sequences of SEQ ID NOs: 145, 146, 147, and 148, respectively. Insome instances, the second binding domain comprises a VH domaincomprising the amino acid sequence of SEQ ID NO: 153. In some instances,the second binding domain further includes at least one (e.g., 1, 2, 3,or 4) of the light chain framework regions FR-L1, FR-L2, FR-L3, andFR-L4 comprising the sequences of SEQ ID NOs: 149, 150, 151, and 152,respectively. In some instances, the second binding domain comprises aVL domain comprising the amino acid sequence of SEQ ID NO: 154. In someinstances, the second binding domain includes a VH domain comprising anamino acid sequence of SEQ ID NOs: 153 and (b) a VL domain comprising anamino acid sequence of SEQ ID NO: 154. Accordingly, in some instances, ahalf-antibody variant of an anti-FcRH5 antibody of the invention may bepaired with a half-antibody variant of anti-CD3 antibody hu40G5c to forman FcRH5 TDB (i.e., an anti-FcRH5/hu40G5c TDB).

In some instances, for example, the invention provides an anti-FcRH5antibody, wherein the second binding domain comprises at least one, two,three, four, five, or six hypervariable regions (HVRs) selected from (a)an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 155; (b) anHVR-H2 comprising the amino acid sequence of SEQ ID NO: 156; (c) anHVR-H3 comprising the amino acid sequence of SEQ ID NO: 157; (d) anHVR-L1 comprising the amino acid sequence of SEQ ID NO: 158; (e) anHVR-L2 comprising the amino acid sequence of SEQ ID NO: 159; and (f) anHVR-L3 comprising the amino acid sequence of SEQ ID NO: 160.

In some instances, the invention provides an anti-FcRH5 antibody,wherein the second binding domain comprises at least one, two, three,four, five, or six hypervariable regions (HVRs) selected from (a) anHVR-H1 comprising the amino acid sequence of SEQ ID NO: 155; (b) anHVR-H2 comprising the amino acid sequence of SEQ ID NO: 162; (c) anHVR-H3 comprising the amino acid sequence of SEQ ID NO: 157; (d) anHVR-L1 comprising the amino acid sequence of SEQ ID NO: 158; (e) anHVR-L2 comprising the amino acid sequence of SEQ ID NO: 159; and (f) anHVR-L3 comprising the amino acid sequence of SEQ ID NO: 160. In someinstances, for example, the second binding domain includes a VH domaincomprising an amino acid sequence having at least 80% (e.g., 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ IDNO: 172 and/or a VL domain comprising an amino acid sequence having atleast 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to, orthe sequence of, SEQ ID NO: 173. In some instances, for example, theanti-FcRH5 antibody includes a second binding domain comprising at leastone (e.g., 1, 2, 3, or 4) of heavy chain framework regions FR-H1, FR-H2,FR-H3, and FR-H4 comprising the sequences of SEQ ID NOs: 164, 165, 166,and 167, respectively. In some instances, the second binding domaincomprises a VH domain comprising the amino acid sequence of SEQ ID NO:172. In some instances, the second binding domain further includes atleast one (e.g., 1, 2, 3, or 4) of the light chain framework regionsFR-L1, FR-L2, FR-L3, and FR-L4 comprising the sequences of SEQ ID NOs:168, 169, 170, and 171, respectively. In some instances, the secondbinding domain comprises a VL domain comprising the amino acid sequenceof SEQ ID NO: 173. In some instances, the second binding domain includesa VH domain comprising an amino acid sequence of SEQ ID NOs: 172 and (b)a VL domain comprising an amino acid sequence of SEQ ID NO: 173.Accordingly, in some instances, a half-antibody variant of an anti-FcRH5antibody of the invention may be paired with a half-antibody variant ofanti-CD3 antibody huUCHT1.v9 to form an FcRH5 TDB (i.e., ananti-FcRH5/huUCHT1.v9 TDB).

In some instances, for example, the invention provides an anti-FcRH5antibody, wherein the binding domain that binds FcRH5 comprises a VHdomain (VH₁) comprising a charged region (CR₁) and a VL domain (VL₁)comprising a charged region (CR₂), wherein the CR, in the VH₁ forms acharge pair with the CR₂ in the VL₁. In some instances, the CR₁comprises a basic amino acid residue and the CR₂ comprises an acidicamino acid residue. In some instances, the CR, comprises a Q39Ksubstitution mutation (EU numbering). In some instances, the CR,consists of the Q39K substitution mutation. In some instances, the CR₂comprises a Q38E substitution mutation (EU numbering). In someinstances, the CR₂ consists of the Q38E substitution mutation. In someinstances, the second binding domain that binds CD3 comprises a VHdomain (VH₂) comprising a charged region (CR₃) and a VL domain (VL₂)comprising a charged region (CR₄), wherein the CR₄ in the VL₂ forms acharge pair with the CR₃ in the VH₂. In some instances, the CR₄comprises a basic amino acid residue and the CR₃ comprises an acidicamino acid residue. In some instances, the CR₄ comprises a Q38Ksubstitution mutation (EU numbering). In some instances, the CR₄consists of the Q38K substitution mutation. In some instances, the CR₃comprises a Q39E substitution mutation (EU numbering). In someinstances, the CR₃ consists of the Q39E substitution mutation. In someinstances, the VL₁ domain is linked to a light chain constant (CL)domain (CL₁) and the VH₁ is linked to a first heavy chain constant (CH1)domain (CH1₁), wherein the CL₁ comprises a charged region (CR₅) and theCH1₁ comprises a charged region (CR₆), and wherein the CR₅ in the CL₁forms a charge pair with the CR₆ in the CH1₁. In some instances, the CR₅comprises a basic amino acid residue and the CR₆ comprises an acidicresidue. In some instances, the CR₅ comprises a V133K substitutionmutation (EU numbering). In some instances, the CR₅ consists of theV133K substitution mutation. In some instances, the CR₆ comprises aS183E substitution mutation (EU numbering). In some instances, the CR₆consists of the S183E substitution mutation.

In some instances, the invention provides an anti-FcRH5 antibody,wherein the VL₂ domain is linked to a CL domain (CL₂) and the VH₂ islinked to a CH1 domain (CH1₂), wherein the CL₂ comprises a chargedregion (CR₇) and the CH1₂ comprises a charged region (CR₈), and whereinthe CR₈ in the CH1₂ forms a charge pair with the CR₇ in the CL₂. In someinstances, the CR₈ comprises a basic amino acid residue and the CR₇comprises an acidic amino acid residue. In some instances, the CR₈comprises a S183K substitution mutation (EU numbering). In someinstances, the CR₈ consists of the S183K substitution mutation. In someinstances, the CR₇ comprises a V133E substitution mutation (EUnumbering). In some instances, the CR₇ consists of the V133Esubstitution mutation.

In some instances, for example, the invention provides an anti-FcRH5antibody, wherein the VL₂ domain is linked to a CL domain (CL₂) and theVH₂ is linked to a CH1 domain (CH1₂), wherein the CL₂ comprises one ormore mutations at amino acid residues F116, L135, S174, S176, and/orT178 (EU numbering) and the CH1₂ comprises one or more mutations atamino acid residues A141, F170, S181, S183, and/or V185 (EU numbering).In some instances, the CL₂ comprises one or more of the followingsubstitution mutations: F116A, L135V, S174A, S176F, and/or T178V. Insome instances, the CL₂ comprises the following substitution mutations:F116A, L135V, S174A, S176F, and T178V. In some instances, the CH1₂comprises one or more of the following substitution mutations: A141I,F170S, S181M, S183A, and/or V185A. In some instances, the CH1₂ comprisesthe following substitution mutations: A141I, F170S, S181M, S183A, andV185A.

In some instances, the invention provides an anti-FcRH5 antibody,wherein the binding domain that binds FcRH5 comprises a VH domain (VH₁)comprising a charged region (CR₁) and a VL domain (VL₁) comprising acharged region (CR₂), wherein the CR₂ in the VL₁ forms a charge pairwith the CR₁ in the VH₁. In some instances, the CR₂ comprises a basicamino acid residue and the CR₁ comprises an acidic amino acid residue.In some instances, the CR₂ comprises a Q38K substitution mutation (EUnumbering). In some instances, the CR₂ consists of the Q38K substitutionmutation. In some instances, the CR₁ comprises a Q39E substitutionmutation (EU numbering). In some instances, the CR₁ consists of the Q39Esubstitution mutation. In some instances, the second binding domain thatbinds CD3 comprises a VH domain (VH₂) comprising a charged region (CR₃)and a VL domain (VL₂) comprising a charged region (CR₄), wherein the CR₃in the VH₂ forms a charge pair with the CR₄ in the VL₂. In someinstances, the CR₃ comprises a basic amino acid residue and the CR₄comprises an acidic amino acid residue. In some instances, the CR₃comprises a Q39K substitution mutation (EU numbering). In someinstances, the CR₃ consists of the Q39K substitution mutation. In someinstances, the CR₄ comprises a Q38E substitution mutation (EUnumbering). In some instances, the CR₄ consists of the Q38E substitutionmutation. In some instances, the VL₁ domain is linked to a light chainconstant (CL) domain (CL₁) and the VH₁ is linked to a first heavy chainconstant (CH1) domain (CH1₁), wherein the CL₁ comprises a charged region(CR₅) and the CH1₁ comprises a charged region (CR₆), and wherein the CR₆in the CH1 forms a charge pair with the CR₅ in the CL₁. In someinstances, the CR₆ comprises a basic amino acid residue and the CR₅comprises an acidic amino acid residue. In some instances, the CR₆comprises a S183K substitution mutation (EU numbering). In someinstances, the CR₆ consists of the S183K substitution mutation. In someinstances, the CR₅ comprises a V133E substitution mutation (EUnumbering). In some instances, the CR₅ consists of the V133Esubstitution mutation.

In some instances, for example, the invention provides an anti-FcRH5antibody, wherein the VL₂ domain is linked to a CL domain (CL₂) and theVH₂ is linked to a CH1 domain (CH1₂), wherein the CL₂ comprises acharged region (CR₇) and the CH1₂ comprises a charged region (CR₈), andwherein the CR₇ in the CL₂ forms a charged pair with the CR₈ in theCH1₂. In some instances, the CR₇ comprises a basic amino acid residueand the CR₈ comprises an acidic residue. In some instances, the CR₇comprises a V133K substitution mutation (EU numbering). In someinstances, the CR₇ consists of the V133K substitution mutation. In someinstances, the CR₈ comprises a S183E substitution mutation (EUnumbering). In some instances, the CR₈ consists of the S183Esubstitution mutation.

In some instances, for example, the invention provides an anti-FcRH5antibody, wherein the VL₂ domain is linked to a CL domain (CL₂) and theVH₂ is linked to a CH1 domain (CH1₂), wherein the CL₂ comprises one ormore mutations at amino acid residues F116, L135, S174, S176, and/orT178 (EU numbering) and the CH1₂ comprises one or more mutations atamino acid residues A141, F170, S181, S183, and/or V185 (EU numbering).In some instances, the CL₂ comprises one or more of the followingsubstitution mutations: F116A, L135V, S174A, S176F, and/or T178V. Insome instances, the CL₂ comprises the following substitution mutations:F116A, L135V, S174A, S176F, and T178V. In some instances, the CH1₂comprises one or more of the following substitution mutations: A141I,F170S, S181M, S183A, and/or V185A. In some instances, the CH1₂ comprisesthe following substitution mutations: A141I, F170S, S181M, S183A, andV185A. In some instances, the anti-FcRH5 antibody comprises one or moreheavy chain constant domains, wherein the one or more heavy chainconstant domains are selected from a first CH2 domain (CH2₁), a firstCH3 domain (CH3₁), a second CH2 domain (CH2₂), and a second CH3 domain(CH3₂). In some instances, at least one of the one or more heavy chainconstant domains is paired with another heavy chain constant domain. Insome instances, the CH3₁ and the CH3₂ each comprise a protuberance (P₁)or a cavity (C₁), and the P₁ or the C₁ in the CH3₁ is positionable inthe C₁ or the P₁, respectively, in the CH3₂. In some instances, the CH3₁and the CH3₂ meet at an interface between the P₁ and the C₁. In someinstances, the CH2₁ and the CH2₂ each comprise (P₂) or a cavity (C₂),and the P₂ or the C₂ in the CH2₁ is positionable in the C₂ or the P₂,respectively, in the CH2₂. In some instances, the CH2₁ and the CH2₂ meetat an interface between the P₂ and the C₂.

In another aspect, the invention provides an anti-FcRH5 antibody thatbinds to FcRH5 and CD3, wherein the anti-FcRH5 antibody comprises ananti-FcRH5 arm comprising a first binding domain at least one, two,three, four, five, or six hypervariable regions (HVRs) selected from (a)an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b) anHVR-H2 comprising the amino acid sequence of SEQ ID NO: 8; (c) an HVR-H3comprising the amino acid sequence of SEQ ID NO: 9; (d) an HVR-L1comprising the amino acid sequence of SEQ ID NO: 12; (e) an HVR-L2comprising the amino acid sequence of SEQ ID NO: 16; and (f) an HVR-L3comprising the amino acid sequence of SEQ ID NO: 23; and an anti-CD3 armcomprising a second binding domain comprising at least one, two, three,four, five, or six hypervariable regions (HVRs) selected from: (a) anHVR-H1 comprising the amino acid sequence of SEQ ID NO: 115; (b) anHVR-H2 comprising the amino acid sequence of SEQ ID NO: 116; (c) anHVR-H3 comprising the amino acid sequence of SEQ ID NO:121; (d) anHVR-L1 comprising the amino acid sequence of SEQ ID NO: 118; (e) anHVR-L2 comprising the amino acid sequence of SEQ ID NO: 119; and (f) anHVR-L3 comprising the amino acid sequence of SEQ ID NO: 123; and whereinthe anti-FcRH5 arm and the anti-CD3 arm each comprise an N297Gsubstitution mutation (EU numbering); and wherein the anti-FcRH5 armcomprises a T366W substitution mutation and the anti-CD3 arm comprises aT366S, L368A, and Y407V substitution mutation. In some instances, theanti-FcRH5 antibody comprises an anti-FcRH5 arm comprising a firstbinding domain comprising the six hypervariable regions (HVRs): (a) anHVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b) an HVR-H2comprising the amino acid sequence of SEQ ID NO: 8; (c) an HVR-H3comprising the amino acid sequence of SEQ ID NO: 9; (d) an HVR-L1comprising the amino acid sequence of SEQ ID NO: 12; (e) an HVR-L2comprising the amino acid sequence of SEQ ID NO: 16; and (f) an HVR-L3comprising the amino acid sequence of SEQ ID NO: 23; and an anti-CD3 armcomprising a second binding domain comprising the six hypervariableregions (HVRs): (a) an HVR-H1 comprising the amino acid sequence of SEQID NO: 115; (b) an HVR-H2 comprising the amino acid sequence of SEQ IDNO: 116; (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:121; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 118;(e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 119; and(f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 123; andwherein the anti-FcRH5 arm and the anti-CD3 arm each comprise an N297Gsubstitution mutation (EU numbering); and wherein the anti-FcRH5 armcomprises a T366W substitution mutation and the anti-CD3 arm comprises aT366S, L368A, and Y407V substitution mutation.

In another aspect, the invention provides an anti-FcRH5 antibody thatbinds to FcRH5 and CD3, wherein the anti-FcRH5 antibody comprises ananti-FcRH5 arm comprising a first binding domain comprising at leastone, two, three, four, five, or six hypervariable regions (HVRs)selected from (a) an HVR-H1 comprising the amino acid sequence of SEQ IDNO: 1; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 8;(c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 9; (d) anHVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (e) anHVR-L2 comprising the amino acid sequence of SEQ ID NO: 16; and (f) anHVR-L3 comprising the amino acid sequence of SEQ ID NO: 23; and ananti-CD3 arm comprising a second binding domain comprising at least one,two, three, four, five, or six hypervariable regions (HVRs) selectedfrom: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:115; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 116;(c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121; (d)an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 118; (e) anHVR-L2 comprising the amino acid sequence of SEQ ID NO: 119; and (f) anHVR-L3 comprising the amino acid sequence of SEQ ID NO: 123; and whereinthe anti-FcRH5 arm comprises a light chain comprising Q38E and V133Ksubstitution mutations and a heavy chain comprising Q39K, S183E, andN297G substitution mutations; and wherein the anti-CD3 arm comprises alight chain comprising Q38K and V133E substitution mutations and a heavychain comprising Q39E, S183K, and N297G substitution mutations (EUnumbering). In some instances, the anti-FcRH5 antibody comprises ananti-FcRH5 arm comprising a first binding domain comprising the sixhypervariable regions (HVRs): (a) an HVR-H1 comprising the amino acidsequence of SEQ ID NO: 1; (b) an HVR-H2 comprising the amino acidsequence of SEQ ID NO: 8; (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 9; (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 12; (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 16; and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 23; and an anti-CD3 arm comprising a secondbinding domain comprising the six hypervariable regions (HVRs): (a) anHVR-H1 comprising the amino acid sequence of SEQ ID NO: 115; (b) anHVR-H2 comprising the amino acid sequence of SEQ ID NO: 116; (c) anHVR-H3 comprising the amino acid sequence of SEQ ID NO: 121; (d) anHVR-L1 comprising the amino acid sequence of SEQ ID NO: 118; (e) anHVR-L2 comprising the amino acid sequence of SEQ ID NO: 119; and (f) anHVR-L3 comprising the amino acid sequence of SEQ ID NO: 123; and whereinthe anti-FcRH5 arm comprises a light chain comprising Q38E and V133Ksubstitution mutations and a heavy chain comprising Q39K, S183E, andN297G substitution mutations; and wherein the anti-CD3 arm comprises alight chain comprising Q38K and V133E substitution mutations and a heavychain comprising Q39E, S183K, and N297G substitution mutations (EUnumbering).

In another aspect, the invention provides an anti-FcRH5 antibody thatbinds to FcRH5 and CD3, wherein the anti-FcRH5 antibody comprises: (a)an anti-FcRH5 arm comprising a first binding domain comprising a VHdomain comprising an amino acid sequence having at least 90% (e.g., 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to, orthe sequence of, SEQ ID NO: 104 and a VL domain comprising an amino acidsequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:105, wherein the anti-FcRH5 arm comprises a light chain comprising Q38Eand V133K substitution mutations and a heavy chain comprising Q39K,S183E, and N297G substitution mutations; and (b) an anti-CD3 armcomprising a second binding domain comprising a VH domain comprising anamino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99%) sequence identity to, or the sequence of,SEQ ID NO: 133 and a VL domain comprising an amino acid sequence havingat least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%)sequence identity to, or the sequence of, SEQ ID NO: 134, wherein theanti-CD3 arm comprises a light chain comprising Q38K and V133Esubstitution mutations and a heavy chain comprising Q39E, S183K, andN297G substitution mutations (EU numbering). In some instances, theinvention provides an anti-FcRH5 antibody that binds to FcRH5 and CD3,wherein the anti-FcRH5 antibody comprises: (a) an anti-FcRH5 armcomprising a first binding domain comprising a VH domain comprising anamino acid sequence of SEQ ID NO: 104 and a VL domain comprising anamino acid sequence of SEQ ID NO: 105, wherein the anti-FcRH5 armcomprises a light chain comprising Q38E and V133K substitution mutationsand a heavy chain comprising Q39K, S183E, and N297G substitutionmutations; and (b) an anti-CD3 arm comprising a second binding domaincomprising a VH domain comprising an amino acid sequence of SEQ ID NO:133 and a VL domain comprising an amino acid sequence of SEQ ID NO: 134,wherein the anti-CD3 arm comprises a light chain comprising Q38K andV133E substitution mutations and a heavy chain comprising Q39E, S183K,and N297G substitution mutations (EU numbering).

In another aspect, the invention provides an anti-FcRH5 antibody thatbinds to FcRH5 and CD3, wherein the anti-FcRH5 antibody comprises ananti-FcRH5 arm comprising a first binding domain comprising at leastone, two, three, four, five, or six hypervariable regions (HVRs)selected from (a) an HVR-H1 comprising the amino acid sequence of SEQ IDNO: 1; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 8;(c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 9; (d) anHVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (e) anHVR-L2 comprising the amino acid sequence of SEQ ID NO: 16; and (f) anHVR-L3 comprising the amino acid sequence of SEQ ID NO: 23; and ananti-CD3 arm comprising a second binding domain comprising at least one,two, three, four, five, or six hypervariable regions (HVRs) selectedfrom (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 115;(b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 116; (c)an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121; (d) anHVR-L1 comprising the amino acid sequence of SEQ ID NO: 118; (e) anHVR-L2 comprising the amino acid sequence of SEQ ID NO: 119; and (f) anHVR-L3 comprising the amino acid sequence of SEQ ID NO: 123; and whereinthe anti-FcRH5 arm comprises a light chain comprising Q38K and V133Esubstitution mutations and a heavy chain comprising Q39E, S183K, andN297G substitution mutations; and wherein the anti-CD3 arm comprises alight chain comprising Q38E and V133K substitution mutations and a heavychain comprising Q39K, S183E, and N297G substitution mutations (EUnumbering). In some instances, the anti-FcRH5 antibody comprises ananti-FcRH5 arm comprising a first binding domain comprising the sixhypervariable regions (HVRs): (a) an HVR-H1 comprising the amino acidsequence of SEQ ID NO: 1; (b) an HVR-H2 comprising the amino acidsequence of SEQ ID NO: 8; (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 9; (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 12; (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 16; and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 23; and an anti-CD3 arm comprising a secondbinding domain comprising the six hypervariable regions (HVRs): (a) anHVR-H1 comprising the amino acid sequence of SEQ ID NO: 115; (b) anHVR-H2 comprising the amino acid sequence of SEQ ID NO: 116; (c) anHVR-H3 comprising the amino acid sequence of SEQ ID NO: 121; (d) anHVR-L1 comprising the amino acid sequence of SEQ ID NO: 118; (e) anHVR-L2 comprising the amino acid sequence of SEQ ID NO: 119; and (f) anHVR-L3 comprising the amino acid sequence of SEQ ID NO: 123; and whereinthe anti-FcRH5 arm comprises a light chain comprising Q38K and V133Esubstitution mutations and a heavy chain comprising Q39E, S183K, andN297G substitution mutations; and wherein the anti-CD3 arm comprises alight chain comprising Q38E and V133K substitution mutations and a heavychain comprising Q39K, S183E, and N297G substitution mutations (EUnumbering).

In another aspect, the invention provides the invention provides ananti-FcRH5 antibody that binds to FcRH5 and CD3, wherein the anti-FcRH5antibody comprises: (a) an anti-FcRH5 arm comprising a first bindingdomain comprising a VH domain comprising an amino acid sequence havingat least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%)sequence identity to, or the sequence of, SEQ ID NO: 104 and a VL domaincomprising an amino acid sequence having at least 90% (e.g., 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 105, wherein the anti-FcRH5 arm comprises alight chain comprising Q38K and V133E substitution mutations and a heavychain comprising Q39E, S183K, and N297G substitution mutations; and (b)an anti-CD3 arm comprising a second binding domain comprising a VHdomain comprising an amino acid sequence of SEQ ID NO: 133 and a VLdomain comprising an amino acid sequence of SEQ ID NO: 134, wherein theanti-CD3 arm comprises a light chain comprising Q38E and V133Ksubstitution mutations and a heavy chain comprising Q39K, S183E, andN297G substitution mutations (EU numbering). In some instances, theinvention provides an anti-FcRH5 antibody that binds to FcRH5 and CD3,wherein the anti-FcRH5 antibody comprises: (a) an anti-FcRH5 armcomprising a first binding domain comprising a VH domain comprising anamino acid sequence of SEQ ID NO: 104 and a VL domain comprising anamino acid sequence of SEQ ID NO: 105, wherein the anti-FcRH5 armcomprises a light chain comprising Q38K and V133E substitution mutationsand a heavy chain comprising Q39E, S183K, and N297G substitutionmutations; and (b) an anti-CD3 arm comprising a second binding domaincomprising a VH domain comprising an amino acid sequence of SEQ ID NO:133 and a VL domain comprising an amino acid sequence of SEQ ID NO: 104,wherein the anti-CD3 arm comprises a light chain comprising Q38E andV133K substitution mutations and a heavy chain comprising Q39K, S183E,and N297G substitution mutations (EU numbering).

In another aspect, the invention provides an anti-FcRH5 antibody thatbinds to FcRH5 and CD3, wherein the anti-FcRH5 antibody comprises ananti-FcRH5 arm comprising a first binding domain comprising at leastone, two, three, four, five, or six hypervariable regions (HVRs)selected from (a) an HVR-H1 comprising the amino acid sequence of SEQ IDNO: 1; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 8;(c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 9; (d) anHVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (e) anHVR-L2 comprising the amino acid sequence of SEQ ID NO: 16; and (f) anHVR-L3 comprising the amino acid sequence of SEQ ID NO: 23; and ananti-CD3 arm comprising a second binding domain comprising at least one,two, three, four, five, or six hypervariable regions (HVRs) selectedfrom (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 115;(b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 116; (c)an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121; (d) anHVR-L1 comprising the amino acid sequence of SEQ ID NO: 118; (e) anHVR-L2 comprising the amino acid sequence of SEQ ID NO: 119; and (f) anHVR-L3 comprising the amino acid sequence of SEQ ID NO: 123; and whereinthe anti-FcRH5 arm comprises a light chain comprising Q38E and V133Ksubstitution mutations and a heavy chain comprising Q39K, S183E, andN297G substitution mutations; and wherein the anti-CD3 arm comprises alight chain comprising Q38K, F116A, L135V, S174A, S176F, and T178Vsubstitution mutations and a heavy chain comprising Q39E, A141I, F170S,S181M, S183A, V185A, and N297G substitution mutations (EU numbering). Insome instances, the anti-FcRH5 antibody comprises an anti-FcRH5 armcomprising a first binding domain comprising the six hypervariableregions (HVRs): (a) an HVR-H1 comprising the amino acid sequence of SEQID NO: 1; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:8; (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 9; (d)an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (e) anHVR-L2 comprising the amino acid sequence of SEQ ID NO: 16; and (f) anHVR-L3 comprising the amino acid sequence of SEQ ID NO: 23; and ananti-CD3 arm comprising a second binding domain comprising the sixhypervariable regions (HVRs): (a) an HVR-H1 comprising the amino acidsequence of SEQ ID NO: 115; (b) an HVR-H2 comprising the amino acidsequence of SEQ ID NO: 116; (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 121; (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 118; (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 119; and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 123; and wherein the anti-FcRH5 arm comprises alight chain comprising Q38E and V133K substitution mutations and a heavychain comprising Q39K, S183E, and N297G substitution mutations; andwherein the anti-CD3 arm comprises a light chain comprising Q38K, F116A,L135V, S174A, S176F, and T178V substitution mutations and a heavy chaincomprising Q39E, A141I, F170S, S181M, S183A, V185A, and N297Gsubstitution mutations (EU numbering).

In another aspect, the invention provides the invention provides ananti-FcRH5 antibody that binds to FcRH5 and CD3, wherein the anti-FcRH5antibody comprises: (a) an anti-FcRH5 arm comprising a first bindingdomain comprising a VH domain comprising an amino acid sequence havingat least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%)sequence identity to, or the sequence of, SEQ ID NO: 104 and a VL domaincomprising an amino acid sequence having at least 90% (e.g., 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 105, wherein the anti-CD3 arm comprises a lightchain comprising Q38K, F116A, L135V, S174A, S176F, and T178Vsubstitution mutations and a heavy chain comprising Q39E, A141I, F170S,5181M, S183A, V185A, and N297G substitution mutations (EU numbering). Insome instances, the invention provides an anti-FcRH5 antibody that bindsto FcRH5 and CD3, wherein the anti-FcRH5 antibody comprises: (a) ananti-FcRH5 arm comprising a first binding domain comprising a VH domaincomprising an amino acid sequence of SEQ ID NO: 104 and a VL domaincomprising an amino acid sequence of SEQ ID NO: 105, wherein theanti-FcRH5 arm comprises a light chain comprising Q38E and V133Ksubstitution mutations and a heavy chain comprising Q39K, S183E, andN297G substitution mutations; and (b) an anti-CD3 arm comprising asecond binding domain comprising a VH domain comprising an amino acidsequence of SEQ ID NO: 133 and a VL domain comprising an amino acidsequence of SEQ ID NO: 134, wherein the anti-CD3 arm comprises a lightchain comprising Q38K, F116A, L135V, S174A, S176F, and T178Vsubstitution mutations and a heavy chain comprising Q39E, A141I, F170S,5181M, S183A, V185A, and N297G substitution mutations (EU numbering).

In another aspect, the invention provides an anti-FcRH5 antibody thatbinds to FcRH5 and CD3, wherein the anti-FcRH5 antibody comprises ananti-FcRH5 arm comprising a first binding domain comprising at leastone, two, three, four, five, or six hypervariable regions (HVRs)selected from (a) an HVR-H1 comprising the amino acid sequence of SEQ IDNO: 1; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 8;(c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 9; (d) anHVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (e) anHVR-L2 comprising the amino acid sequence of SEQ ID NO: 16; and (f) anHVR-L3 comprising the amino acid sequence of SEQ ID NO: 23; and ananti-CD3 arm comprising a second binding domain comprising at least one,two, three, four, five, or six hypervariable regions (HVRs) selectedfrom (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 115;(b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 116; (c)an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121; (d) anHVR-L1 comprising the amino acid sequence of SEQ ID NO: 118; (e) anHVR-L2 comprising the amino acid sequence of SEQ ID NO: 119; and (f) anHVR-L3 comprising the amino acid sequence of SEQ ID NO: 123; and whereinthe anti-FcRH5 arm comprises a light chain comprising Q38K and V133Esubstitution mutations and a heavy chain comprising Q39E, S183K, andN297G substitution mutations; and wherein the anti-CD3 arm comprises alight chain comprising Q38E, F116A, L135V, S174A, S176F, and T178Vsubstitution mutations and a heavy chain comprising Q39K, A141I, F170S,S181M, S183A, V185A, and N297G substitution mutations (EU numbering). Insome instances, the anti-FcRH5 antibody comprises an anti-FcRH5 armcomprising a first binding domain comprising the six hypervariableregions (HVRs): (a) an HVR-H1 comprising the amino acid sequence of SEQID NO: 1; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:8; (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 9; (d)an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (e) anHVR-L2 comprising the amino acid sequence of SEQ ID NO: 16; and (f) anHVR-L3 comprising the amino acid sequence of SEQ ID NO: 23; and ananti-CD3 arm comprising a second binding domain comprising the sixhypervariable regions (HVRs): (a) an HVR-H1 comprising the amino acidsequence of SEQ ID NO: 115; (b) an HVR-H2 comprising the amino acidsequence of SEQ ID NO: 116; (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 121; (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 118; (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 119; and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 123; and wherein the anti-FcRH5 arm comprises alight chain comprising Q38K and V133E substitution mutations and a heavychain comprising Q39E, S183K, and N297G substitution mutations; andwherein the anti-CD3 arm comprises a light chain comprising Q38E, F116A,L135V, S174A, S176F, and T178V substitution mutations and a heavy chaincomprising Q39K, A141I, F170S, 5181M, S183A, V185A, and N297Gsubstitution mutations (EU numbering).

In another aspect, the invention provides an anti-FcRH5 antibody thatbinds to FcRH5 and CD3, wherein the anti-FcRH5 antibody comprises: (a)an anti-FcRH5 arm comprising a first binding domain comprising a VHdomain comprising an amino acid sequence having at least 90% (e.g., 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to, orthe sequence of, SEQ ID NO: 104 and a VL domain comprising an amino acidsequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:105, wherein the anti-FcRH5 arm comprises a light chain comprising Q38Kand V133E substitution mutations and a heavy chain comprising Q39E,S183K, and N297G substitution mutations; and (b) an anti-CD3 armcomprising a second binding domain comprising a VH domain comprising anamino acid sequence of SEQ ID NO: 133 and a VL domain comprising anamino acid sequence of SEQ ID NO: 134, wherein the anti-CD3 armcomprises a light chain comprising Q38E, F116A, L135V, S174A, S176F, andT178V substitution mutations and a heavy chain comprising Q39K, A141I,F170S, S181M, S183A, V185A, and N297G substitution mutations (EUnumbering). In some instances, the invention provides an anti-FcRH5antibody that binds to FcRH5 and CD3, wherein the anti-FcRH5 antibodycomprises: (a) an anti-FcRH5 arm comprising a first binding domaincomprising a VH domain comprising an amino acid sequence of SEQ ID NO:104 and a VL domain comprising an amino acid sequence of SEQ ID NO: 105,wherein the anti-FcRH5 arm comprises a light chain comprising Q38K andV133E substitution mutations and a heavy chain comprising Q39E, S183K,and N297G substitution mutations; and (b) an anti-CD3 arm comprising asecond binding domain comprising a VH domain comprising an amino acidsequence of SEQ ID NO:133 and a VL domain comprising an amino acidsequence of SEQ ID NO: 134, wherein the anti-CD3 arm comprises a lightchain comprising Q38E, F116A, L135V, S174A, S176F, and T178Vsubstitution mutations and a heavy chain comprising Q39K, A141I, F170S,S181M, S183A, V185A, and N297G substitution mutations (EU numbering).

7. Antibody Variants

In certain embodiments, amino acid sequence variants of the anti-FcRH5antibodies of the invention (e.g., bispecific anti-FcRH5 antibodies ofthe invention that bind to FcRH5 and a second biological molecule, e.g.,CD3, such as FcRH5 TDB antibodies of the invention or variants thereof)are contemplated. For example, it may be desirable to improve thebinding affinity and/or other biological properties of the antibody.Amino acid sequence variants of an antibody may be prepared byintroducing appropriate modifications into the nucleotide sequenceencoding the antibody, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, for example,antigen-binding.

a. Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the HVRs and FRs. Conservative substitutions areshown in Table 1 under the heading of “preferred substitutions.” Moresubstantial changes are provided in Table 1 under the heading of“exemplary substitutions,” and as further described below in referenceto amino acid side chain classes. Amino acid substitutions may beintroduced into an antibody of interest and the products screened for adesired activity, for example, retained/improved antigen binding,decreased immunogenicity, or improved ADCC or CDC.

TABLE 1 Exemplary and Preferred Amino Acid Substitutions OriginalExemplary Preferred Residue Substitutions Substitutions Ala (A) Val;Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; ArgGln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu(E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I)Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine; Ile; Val;Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile LeuPhe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr ThrThr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser PheVal (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

Amino acids may be grouped according to common side-chain properties:

-   -   (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;    -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;    -   (3) acidic: Asp, Glu;    -   (4) basic: His, Lys, Arg;    -   (5) residues that influence chain orientation: Gly, Pro;    -   (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g., a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antibody and/or will have substantially retainedcertain biological properties of the parent antibody. An exemplarysubstitutional variant is an affinity matured antibody, which may beconveniently generated, e.g., using phage display-based affinitymaturation techniques such as those described herein. Briefly, one ormore HVR residues are mutated and the variant antibodies displayed onphage and screened for a particular biological activity (e.g. bindingaffinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improveantibody affinity. Such alterations may be made in HVR “hotspots,” i.e.,residues encoded by codons that undergo mutation at high frequencyduring the somatic maturation process (see, e.g., Chowdhury, MethodsMol. Biol. 207:179-196, 2008), and/or residues that contact antigen,with the resulting variant VH or VL being tested for binding affinity.Affinity maturation by constructing and reselecting from secondarylibraries has been described, e.g., in Hoogenboom et al. in Methods inMolecular Biology 178:1-37 (O'Brien et al. ed., Human Press, Totowa,N.J., (2001).) In some embodiments of affinity maturation, diversity isintroduced into the variable genes chosen for maturation by any of avariety of methods (e.g., error-prone PCR, chain shuffling, oroligonucleotide-directed mutagenesis). A secondary library is thencreated. The library is then screened to identify any antibody variantswith the desired affinity. Another method to introduce diversityinvolves HVR-directed approaches, in which several HVR residues (e.g.,4-6 residues at a time) are randomized. HVR residues involved in antigenbinding may be specifically identified, e.g., using alanine scanningmutagenesis or modeling. CDR-H3 and CDR-L3 in particular are oftentargeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. Such alterations may, for example, be outside ofantigen contacting residues in the HVRs. In certain embodiments of thevariant VH and VL sequences provided above, each HVR either isunaltered, or contains no more than one, two, or three amino acidsubstitutions.

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham and Wells (1989) Science,244:1081-1085. In this method, a residue or group of target residues(e.g., charged residues such as arg, asp, his, lys, and glu) areidentified and replaced by a neutral or negatively charged amino acid(e.g., alanine or polyalanine) to determine whether the interaction ofthe antibody with antigen is affected. Further substitutions may beintroduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, a crystal structure of an antigen-antibody complex toidentify contact points between the antibody and antigen. Such contactresidues and neighboring residues may be targeted or eliminated ascandidates for substitution. Variants may be screened to determinewhether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g., for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

b. Glycosylation Variants

In certain embodiments, anti-FcRH5 antibodies of the invention can bealtered to increase or decrease the extent to which the antibody isglycosylated. Addition or deletion of glycosylation sites to anti-FcRH5antibody of the invention may be conveniently accomplished by alteringthe amino acid sequence such that one or more glycosylation sites iscreated or removed. Addition or removal of a glycosylation site mayalter the effector function of an antibody, such as an anti-FcRH5antibody (e.g., an FcRH5 TDB). In some embodiments, the anti-FcRH5antibody (e.g., an FcRH5 TDB) may contain an aglycosylation sitemutation. In some embodiments, the aglycosylation site mutation is asubstitution mutation. In some embodiments, the aglycosylation sitemutation reduces the effector function of the anti-FcRH5 antibody by atleast 1% or more (e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, or 90% or more). In some embodiments, thesubstitution mutation is at amino acid residue N297, L234, L235, D265,and/or P329 (EU numbering). In some embodiments, the substitutionmutation is selected from the group consisting of N297G, N297A, L234A,L235A, D265A, and P329G. In some embodiments, the substitution mutationis an N297G mutation.

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright et al. TIBTECH 15:26-32, 1997. Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

In one embodiment, anti-FcRH5 antibody variants are provided having acarbohydrate structure that lacks fucose attached (directly orindirectly) to an Fc region. For example, the amount of fucose in suchantibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from20% to 40%. The amount of fucose is determined by calculating theaverage amount of fucose within the sugar chain at Asn297, relative tothe sum of all glycostructures attached to Asn297 (e. g. complex, hybridand high mannose structures) as measured by MALDI-TOF mass spectrometry,as described in WO 2008/077546, for example. Asn297 refers to theasparagine residue located at about position 297 in the Fc region (EUnumbering of Fc region residues); however, Asn297 may also be locatedabout ±3 amino acids upstream or downstream of position 297, i.e.,between positions 294 and 300, due to minor sequence variations inantibodies. Such fucosylation variants may have improved ADCC function.See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publicationsrelated to “defucosylated” or “fucose-deficient” antibody variantsinclude: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614;US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki etal. J. Mol. Biol. 336:1239-1249, 2004; Yamane-Ohnuki et al. Biotech.Bioeng. 87: 614, 2004. Examples of cell lines capable of producingdefucosylated antibodies include Lecl3 CHO cells deficient in proteinfucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545, 1986; USPat Appl No US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adamset al. especially at Example 11), and knockout cell lines, such asalpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g.,Yamane-Ohnuki et al. Biotech. Bioeng. 87:614, 2004; Kanda, Y. et al.Biotechnol. Bioeng., 94(4):680-688, 2006; and WO2003/085107).

Anti-FcRH5 antibodies variants are further provided with bisectedoligosaccharides, for example, in which a biantennary oligosaccharideattached to the Fc region of the antibody is bisected by GlcNAc. Suchantibody variants may have reduced fucosylation and/or improved ADCCfunction. Examples of such antibody variants are described, e.g., in WO2003/011878 (Jean-Mairet et al.); U.S. Pat. No. 6,602,684 (Umana etal.); and US 2005/0123546 (Umana et al.). Antibody variants with atleast one galactose residue in the oligosaccharide attached to the Fcregion are also provided. Such antibody variants may have improved CDCfunction. Such antibody variants are described, e.g., in WO 1997/30087(Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).

c. VH, VL, CH1, and CL Domain Variants

In certain embodiments, one or more amino acid modifications may beintroduced into the heavy chain variable (VH, e.g., VH₁ and/or VH₂)domain, light chain variable (VL, e.g., VL₁ and/or VL₂) domain, heavychain constant (CH1, e.g., CH1₁ and/or CH1₂) domain, and/or the lightchain constant (CL, e.g., CL₁ and/or CL₂) domain of an anti-FcRH5antibody of the invention (e.g., a bispecific anti-FcRH5 antibody of theinvention that binds to FcRH5 and a second biological molecule, e.g., anFcRH5 TDB antibody of the invention or variant thereof). The VH, VL,CH1, and/or CL domains may have amino acid modifications (e.g.,substitutions) at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10)amino acid positions. In particular embodiments, the amino acidsubstitutions comprise acidic and/or basic amino acid residues. Aminoacid modifications, may form charged regions within the VH, VL, CH1,and/or CL domains (e.g., regions comprising acidic and/or basic aminoacid residues). Charged regions within the VH, VL, CH1, and/or CLdomains may interact with a second charged regions of opposite overallcharge to form a charge pair. For example, amino acid modifications maymediate the formation of a charge pair between charged regions presentwithin the VL₁ and VH₁ domains of the FcRH5 arm of a TDB. In someinstances, amino acid modifications may mediate the formation of acharge pair between charged regions present within the CL₁ and CH1₁domains of the FcRH5 arm of a TDB. In some instances, amino acidmodifications may mediate the formation of a charge pair between chargedregions present within the VL₂ and VH₂ domains of the second arm of aTDB (e.g., the CD3 arm of the TDB). In some instances, amino acidmodifications may mediate the formation of a charge pair between chargedregions present within the CL₂ and CH1₂ domains of the second arm of aTDB (e.g., the CD3 arm of the TDB). Exemplary configurations of FcRH5TDBs containing VH, VL, CH1, and/or CL domain variants are presented inFIGS. 1A-1S.

In certain embodiments, an anti-FcRH5 antibody (e.g., a bispecificanti-FcRH5 antibody of the invention that binds to FcRH5 and a secondbiological molecule, e.g., CD3, such as an FcRH5 TDB antibody of theinvention) comprises one or more asymmetrical modifications in the VH,VL, CH1, and/or CL regions to facilitate correct heavy/light chainpairing. In other embodiments, the anti-FcRH5 antibody further comprisesone or more modifications in the Fc region to facilitateheterodimerization of the two arms (e.g., an anti-FcRH5 arm and ananti-CD3 arm) of the anti-FcRH5 antibody (e.g., FcRH5 TDB).

In some embodiments, the CH1₁ domain comprises an amino acidsubstitution at S183 (EU numbering) and the CL₁ domain comprises anamino acid substitution at V133 (EU numbering). In other embodiments,the light chain of the first arm (e.g., an FcRH5 binding arm) of theanti-FcRH5 antibody is a kappa chain. In some embodiments, the lightchain of the second arm (e.g., an anti-CD3 binding arm) of theanti-FcRH5 antibody is a kappa chain. In certain embodiments, the lightchains in both arms of the anti-FcRH5 antibody (e.g, the FcRH5 bindingarm and the CD3 binding arm) are kappa chains.

In some embodiments, the CH1 domain comprises an S183E mutation and theCL₁ domain comprises a V133K mutation. In other embodiments, the CH1domain comprises an S183K mutation and the CL₁ domain comprises a V133Emutation.

In some embodiments, the CH1₁ domain comprises an S183K mutation, theCL₁ domain comprises a V133E mutation, the CH1₂ domain comprises anS183E mutation, and the CL₂ domain comprises a V133K mutation.

In some embodiments, the anti-FcRH5 antibody further comprises a Q39Emutation in the VH₁ domain, a Q38K mutation in the VL₁ domain, a Q39Kmutation in the VH₂ domain, and a Q38E mutation in the VL₂ domain.

In some embodiments, the anti-FcRH5 antibody further comprises a knob(e.g., protuberance) mutation in the CH3₁ domain and a hole (e.g.,cavity) mutation in the CH3₂ domain. In certain embodiments, the knobmutation comprises a T366W mutation (EU numbering). In certainembodiments, the hole mutation comprises at least one, at least two, orall three of T366S, L368A, and Y407V mutations (EU numbering). Incertain embodiments, the anti-FcRH5 antibody further comprises a T366Wmutation in the first heavy chain and T366S, L368A, and Y407V mutationsin the second heavy chain.

In some embodiments, the CH1₁ domain comprises A141I, F170S, S181M,S183A, and V185A mutations and the CL₁ domain comprises F116A, L135V,S174A, S176F, and T178V mutations. In certain other embodiments, theCH1₁ domain comprises A141I, F170S, S181M, S183A, and V185A mutations;the CL₁ domain comprises F116A, L135V, S174A, S176F, and T178Vmutations; the CH1₂ domain comprises an S183E mutation; and the CL₂domain comprises a V133K mutation.

d. Fc Region Variants

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an anti-FcRH5 antibody of the invention(e.g., a bispecific anti-FcRH5 antibody of the invention that binds toFcRH5 and a second biological molecule, e.g., CD3, such as a TDBantibody of the invention or variant thereof), thereby generating an Fcregion variant (see e.g., U.S. Pub. No. 2012/0251531, which isincorporated herein by reference in its entirety). In certain otherembodiments, an anti-FcRH5 antibody comprises one or more modificationsin the Fc region to facilitate heterodimerization of the two arms (e.g.,an anti-FcRH5 arm and an anti-CD3 arm) of the anti-FcRH5 antibody (e.g.,FcRH5 TDB). In certain embodiments, the anti-FcRH5 antibody (e.g., FcRH5TDB) having one or more Fc modifications may also have one or moremodifications in the VH, VL, CH1, and/or CL domains, as described above.In some embodiments, the FcRH5 antibody (e.g., FcRH5 TDB) containing Fc,VH, VL, CH1, and/or CL modifications may be produced by a one-cellantibody production approach, as described herein. The Fc region variantmay comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3or IgG4 Fc region) comprising an amino acid modification (e.g., asubstitution) at one or more amino acid positions.

In certain embodiments, the invention contemplates an anti-FcRH5antibody variant that possesses some but not all effector functions,which make it a desirable candidate for applications in which thehalf-life of the antibody in vivo is important yet certain effectorfunctions (such as complement and ADCC) are unnecessary or deleterious.In vitro and/or in vivo cytotoxicity assays can be conducted to confirmthe reduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express Fc RII only, whereas monocytes express Fc RI, Fc RII, andFc RIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch et al. Annu. Rev. Immunol. 9:457-492, 1991.Non-limiting examples of in vitro assays to assess ADCC activity of amolecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g.,Hellstrom et al. Proc. NatlAcad. Sci. USA 83:7059-7063, 1986) andHellstrom et al. Proc. NatlAcad. Sci. USA 82:1499-1502, 1985; 5,821,337(see Bruggemann, et al. J. Exp. Med. 166:1351-1361, 1987).Alternatively, non-radioactive assays methods may be employed (see, forexample, ACTI™ non-radioactive cytotoxicity assay for flow cytometry(CellTechnology, Inc. Mountain View, Calif.; and CYTOTOX 96®non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g., in a animal model such as that disclosed in Clynes et al.Proc. NatlAcad. Sci. USA 95:652-656, 1998. Clq binding assays may alsobe carried out to confirm that the antibody is unable to bind Clq andhence lacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO2006/029879 and WO 2005/100402. To assess complement activation, a CDCassay may be performed (see, for example, Gazzano-Santoro et al. J.Immunol. Methods 202:163, 1996; Cragg, M. S. et al. Blood.101:1045-1052, 2003; and Cragg, M. S. and M. J. Glennie Blood.103:2738-2743, 2004). FcRn binding and in vivo clearance/half lifedeterminations can also be performed using methods known in the art(see, e.g., Petkova, S. B. et al. Int'l. Immunol. 18(12):1759-1769,2006).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. Nos. 6,737,056 and 8,219,149). Such Fcmutants include Fc mutants with substitutions at two or more of aminoacid positions 265, 269, 270, 297 and 327, including the so-called“DANA” Fc mutant with substitution of residues 265 and 297 to alanine(U.S. Pat. Nos. 7,332,581 and 8,219,149).

In certain embodiments, the proline at position 329 of a wild-type humanFc region in the antibody is substituted with glycine or arginine or anamino acid residue large enough to destroy the proline sandwich withinthe Fc/Fc.gamma. receptor interface that is formed between the proline329 of the Fc and tryptophan residues Trp87 and Trp110 of FcgRIII(Sondermann et al. Nature. 406:267-273, 2000). In certain embodiments,the antibody comprises at least one further amino acid substitution. Inone embodiment, the further amino acid substitution is S228P, E233P,L234A, L235A, L235E, N297A, N297D, or P331S, and still in anotherembodiment the at least one further amino acid substitution is L234A andL235A of the human IgG1 Fc region or S228P and L235E of the human IgG4Fc region (see e.g., US 2012/0251531), and still in another embodimentthe at least one further amino acid substitution is L234A and L235A andP329G of the human IgG1 Fc region.

Certain antibody variants with improved or diminished binding to FcRsare described (see, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields et al. J. Biol. Chem. 9(2):6591-6604, 2001).

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions which improve ADCC, e.g.,substitutions at positions 298, 333, and/or 334 of the Fc region (EUnumbering of residues).

In some embodiments, alterations are made in the Fc region that resultin altered (i.e., either improved or diminished) Clq binding and/orComplement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat.No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164:4178-4184, 2000.

Antibodies with increased half-lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al. J. Immunol. 117:587, 1976; Kimet al. J. Immunol. 24:249, 1994), are described in US2005/0014934A1(Hinton et al.). Those antibodies comprise an Fc region with one or moresubstitutions therein which improve binding of the Fc region to FcRn.Such Fc variants include those with substitutions at one or more of Fcregion residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317,340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g.,substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).

See also Duncan et al. Nature 322:738-40, 1988; U.S. Pat. Nos.5,648,260; 5,624,821; and WO 94/29351 concerning other examples of Fcregion variants.

In certain embodiments, the anti-FcRH5 antibody comprises anaglycosylation site mutation. The aglycosylation site mutation may be asubstitution mutation. The aglycosylation site mutation may reduceeffector function of the anti-FcRH5 antibody, as compared to anunmutated version, by at least 1% (e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) or more. In someinstances, the substitution mutation is at amino acid residue N297,L234, L235, D265, and/or P329 (EU numbering). Additionally, thesubstitution mutation may be selected from the group consisting ofN297G, N297A, L234A, L235A, D265A, and P329G. In particular instances,the substitution mutation is an N297G mutation.

e. Knob and Hole Variants

Knob and hole variants of antibodies may also be produced in which anamino acid modification (e.g., a substitution) of one or more (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid residues creates aprotuberance (knob) or a cavity (hole). In some instances, theprotuberance located on one polypeptide of the antibody is positionablewithin the cavity located on a second polypeptide of the antibody suchthat the two constituent polypeptides of the antibody can meet at aninterface between the protuberance and the cavity. A knob may be formedby the substitution of one or more amino acid residues with one or moreamino acid residues of larger size. A cavity may be formed by thesubstitution of a one or more amino acid residues with one or more aminoacid residues of smaller size. The protuberance or cavity can beintroduced into either arm of an anti-FcRH5 antibody (e.g., an FcRH5 TDB(e.g., either the anti-FcRH5 arm or the anti-CD3 arm)). Knob and holemodifications (e.g., of the Fc domain) are particularly useful inincreasing overall yield, homogeneity, and stability of bispecificantibodies (e.g., TDBs). In some instances (e.g., when theprotuberance-cavity pair are located on the LC/HC interfaces (e.g., VHand VL or CL and CH1)), amino acid modifications that introduce knob andhole modifications can reduce light chain swapping. In some embodiments,the protuberance is formed by introducing a T366W mutation into one armof an anti-FcRH5 antibody (e.g., either the anti-FcRH5 arm or theanti-CD3 arm). In some embodiments, the T366W mutation is in theanti-FcRH5 arm. In some embodiments the T366W mutation is in theanti-CD3 arm. In some embodiments, a cavity is formed by introducing aT366W, L368A, and/or a Y407V mutation into one arm of an anti-FcRH5antibody (e.g., either the anti-FcRH5 arm or the anti-CD3 arm). In someembodiments, a T366W, L368A, and/or a Y407V mutation is in theanti-FcRH5 arm. In some embodiments, a T366W, L368A, and/or a Y407Vmutation is in the anti-CD3 arm.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein andCuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al.,EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g.,U.S. Pat. No. 5,731,168). “Knob-in-hole” engineering of multispecificantibodies may be utilized to generate a first arm containing a knob anda second arm containing the hole into which the knob of the first armmay bind. For example, the TDB antibodies of the invention may contain aknob located on its anti-CD3 arm and a hole located on itstumor-targeting arm. Alternatively, the TDB antibodies of the inventionmay contain a knob located on its tumor-targeting arm and a hole locatedon its anti-CD3 arm. Multispecific antibodies may also be engineeredusing immunoglobulin crossover (also known as Fab domain exchange orCrossMab format) technology (see eg., WO2009/080253; Schaefer et al.,Proc. Natl. Acad. Sci. USA, 108:11187-11192 (2011)). Multispecificantibodies may also be made by engineering electrostatic steeringeffects for making antibody Fc-heterodimeric molecules (WO2009/089004A1); cross-linking two or more antibodies or fragments (see,e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229: 81(1985)); using leucine zippers to produce bi-specific antibodies (see,e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using“diabody” technology for making bispecific antibody fragments (see,e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448(1993)); and using single-chain Fv (sFv) dimers (see, e.g. Gruber etal., J. Immunol., 152:5368 (1994)); and preparing trispecific antibodiesas described, e.g., in Tutt et al. J. Immunol. 147: 60 (1991).

f. Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., “thioMAbs,” in which one or more residuesof an antibody are substituted with cysteine residues. In particularembodiments, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such as drugmoieties or linker-drug moieties, to create an immunoconjugate, asdescribed further herein. In certain embodiments, any one or more of thefollowing residues may be substituted with cysteine: V205 (Kabatnumbering) of the light chain; A118 (EU numbering) of the heavy chain;and S400 (EU numbering) of the heavy chain Fc region. Cysteineengineered antibodies may be generated as described, for example, inU.S. Pat. No. 7,521,541, which is incorporated herein by reference inits entirety.

g. Other Antibody Derivatives

In certain embodiments, an anti-FcRH5 antibody of the invention (e.g.,bispecific anti-FcRH5 antibody of the invention that binds to FcRH5 anda second biological molecule, e.g., CD3, such as a TDB antibody of theinvention or variant thereof) provided herein may be further modified tocontain additional nonproteinaceous moieties that are known in the artand readily available. The moieties suitable for derivatization of theantibody include but are not limited to water soluble polymers.Non-limiting examples of water soluble polymers include, but are notlimited to, polyethylene glycol (PEG), copolymers of ethyleneglycol/propylene glycol, carboxymethylcellulose, dextran, polyvinylalcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane,ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymersor random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer areattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions,etc.

In another embodiment, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the nonproteinaceous moiety is a carbonnanotube (Kam et al. Proc. Natl. Acad. Sci. USA 102:11600-11605, 2005).The radiation may be of any wavelength, and includes, but is not limitedto, wavelengths that do not harm ordinary cells, but which heat thenonproteinaceous moiety to a temperature at which cells proximal to theantibody-nonproteinaceous moiety are killed.

B. Recombinant Methods and Compositions

Anti-FcRH5 antibodies of the invention (e.g., bispecific anti-FcRH5antibodies of the invention that bind to FcRH5 and a second biologicalmolecule, e.g., CD3, such as TDB antibodies of the invention or variantsthereof) may be produced using recombinant methods and compositions, forexample, as described in U.S. Pat. No. 4,816,567. In one embodiment,isolated nucleic acid encoding an anti-FcRH5 antibody described hereinis provided. In one embodiment, the host cell is eukaryotic, e.g., aChinese Hamster Ovary (CHO) cell. In one embodiment, the host cell isprokaryotic, e.g., an E. coli cell. In one embodiment, a method ofmaking an anti-FcRH5 antibody is provided, wherein the method comprisesculturing a host cell comprising a nucleic acid encoding the antibody,as provided above, under conditions suitable for expression of theantibody, and optionally recovering the antibody from the host cell (orhost cell culture medium). In another embodiment, the method furthercomprises culturing a second host cell comprising a second nucleic acidencoding an anti-CD3 antibody that comprises a binding domain that bindsCD3. In some embodiments, the host cells are co-cultured. A furtherembodiment, comprises recovering the bispecific anti-FcRH5 antibody fromthe host cell or the culture medium. In some embodiments, the anti-FcRH5and anti-CD3 antibodies are produced in the same host cell (e.g., aone-cell approach). In some embodiments, the anti-FcRH5 and anti-CD3antibodies are produced in separate host cells (e.g., a two-cellapproach).

In the one-cell and two-cell approaches, one or more plasmids encodingthe FcRH5 TDB (e.g, an FcRH5 half-antibody and a CD3 half-antibody) areintroduced into one or more host cells for culture and expression of theTDB. In one instance, a single plasmid may encode both the FcRH5half-antibody and the CD3 half-antibody. Alternatively, thehalf-antibodies can be encoded by separate plasmids. In anotherinstance, the heavy chain of each half-antibody is encoded on a firstplasmid, while the light chain of each half-antibody is encoded on asecond plasmid. In the one-cell approach, the FcRH5 TDB is produced in asingle host. In the two-cell approach, the FcRH5 TDB is produced byexpressing the half-antibodies in separate hosts (e.g., separatecultures of the same host cells, or separate cultures of different hostcells). In the two-cell approach, the two hosts can be cultured in thesame vessel or in different vessels. The two host cultures can becombined prior to lysis and purification of the FcRH5 TDB or the twohalf-antibodies can be purified separately.

In some embodiments, an anti-FcRH5 antibody of the invention (e.g., abispecific anti-FcRH5 antibody of the invention that binds to FcRH5 anda second biological molecule, e.g., CD3, such as an FcRH5 TDB antibodyof the invention) that has been modified to include asymmetricalmodifications (e.g., mutations of the VH, VL, CH1, CL and/or Fc domainsdescribed above) is produced using a one-cell approach, which results inimproved correct heavy chain/light chain pairing and/or improved yieldof the anti-FcRH5 antibody as compared to an anti-FcRH5 antibody thathas not been modified to include the asymmetrical modifications.

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.Nos. 5,648,237, 5,789,199, and 5,840,523 (see, e.g., Charlton, Methodsin Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J., 2003), pp. 245-254, describing expression of antibody fragments inE. coli.) After expression, the antibody may be isolated from thebacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross, Nat. Biotech. 22:1409-1414, 2004, and Li et al.Nat. Biotech. 24:210-215, 2006.

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham et al. J. Gen Virol. 36:59, 1977);baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather Biol. Reprod. 23:243-251, 1980); monkeykidney cells (CV1); African green monkey kidney cells (VERO-76); humancervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo ratliver cells (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., inMather et al. Annals N.Y. Acad. Sci. 383:44-68, 1982; MRC-5 cells; andFS4 cells. Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al. Proc.Natl. Acad. Sci. USA 77:4216, 1980); and myeloma cell lines such as YO,NSO and Sp2/0. For a review of certain mammalian host cell linessuitable for antibody production, see, e.g., Yazaki et al. Methods inMolecular Biology, 248:255-268, 2003.

C. Assays

Anti-FcRH5 antibodies of the invention (e.g., bispecific anti-FcRH5antibodies of the invention that bind to FcRH5 and a second biologicalmolecule, e.g., CD3, such as TDB antibodies of the invention or variantsthereof) provided herein may be identified, screened for, orcharacterized for their physical/chemical properties and/or biologicalactivities by various assays known in the art.

1. Binding Assays and Other Assays

In one aspect, an anti-FcRH5 antibody of the invention is tested for itsantigen binding activity, for example, by known methods such as ELISA,Western blot, etc.

In another aspect, competition assays may be used to identify anantibody that competes with an anti-FcRH5 antibody of the invention forbinding to FcRH5.

In an exemplary competition assay, immobilized FcRH5 is incubated in asolution comprising a first labeled antibody that binds to FcRH5 and asecond unlabeled antibody that is being tested for its ability tocompete with the first antibody for binding to FcRH5. The secondantibody may be present in a hybridoma supernatant. As a control,immobilized FcRH5 is incubated in a solution comprising the firstlabeled antibody but not the second unlabeled antibody. After incubationunder conditions permissive for binding of the first antibody to FcRH5,excess unbound antibody is removed, and the amount of label associatedwith immobilized FcRH5 is measured. If the amount of label associatedwith immobilized FcRH5 is substantially reduced in the test samplerelative to the control sample, then that indicates that the secondantibody is competing with the first antibody for binding to FcRH5. See,e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual. Ch.14(Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

2. Activity Assays

In one aspect, assays are provided for identifying anti-FcRH5 antibodiesthereof having biological activity. Biological activity may include, forexample, binding to FcRH5, or a peptide fragment thereof, either invivo, in vitro, or ex vivo. In the case of a multispecific (e.g.,bispecific) anti-FcRH5 antibody of the invention (e.g., a TDB antibodyhaving one anti-FcRH5 arm and one arm that recognizes a secondbiological molecule, e.g., CD3), biological activity may also include,for example, effector cell activation (e.g., T cell (e.g., CD8+ and/orCD4+ T cell) activation), effector cell population expansion (i.e., anincrease in T cell count), target cell population reduction (i.e., adecrease in the population of cells expressing the second biologicalmolecule on their cell surfaces), and/or target cell killing. Antibodieshaving such biological activity in vivo and/or in vitro are provided. Incertain embodiments, an antibody of the invention is tested for suchbiological activity, as described in detail in the Examples hereinbelow.

In some embodiments, the anti-FcRH5 antibody (e.g., FcRH5 TDB) activitycomprises ability to support B cell killing and/or the activation of thecytotoxic T cells. In certain embodiments, an FcRH5 TDB antibody of theinvention is tested for such B cell killing and/or the activation of thecytotoxic effect of T cells biological activity by any of the methodsdescribed herein, in particular the Examples. In some embodiments of anyof these activity assays, PBMCs may be isolated from whole blood ofhealthy donors by Ficoll separation. In particular, human blood may becollected in heparinized syringes, and PBMCs isolated using Leucosep andFicoll Paque Plus. If needed CD4+T and CD8+ T cells may be separatedwith Miltenyi kits according to manufacturer's instructions.

Further, cells may be washed in RPMI medium containing 10% FBS,supplemented with GlutaMax, penicillin & streptomycin, and 0.2 millionsuspended cells added to a 96-well U-bottom plate. Cells may be culturedin RPMI1640 supplemented with 10% FBS at 37° C. in a humidified standardcell culture incubator. For BJAB cell killing assays, 20,000 BJAB cellsmay be incubated with effector cells, either as huPBMCs or purified Tcells, as indicated ratios per assay, in the presence of variousconcentrations of TDB antibodies for 24 hours. For endogenous B cellkilling assays, 200,000 huPBMCs may be incubated with variousconcentrations of TDB antibodies for 24 hours.

After culturing, cells may be washed with FACS buffer (0.5% BSA, 0.05%Na Azide in PBS). Cells may then be stained in FACS buffer, washed withFACS buffer and suspended in 100μl of FACS buffer containing 1 μg/mlPropidium Iodide. Data may be collected on a FACSCalibur flow cytometerand analyzed using FlowJo. Live B cells may be gated out as PI-negativeCD19+ or PI-negative CD20+ B cells by FACS, and absolute cell count maybe obtained with FITC beads added to reaction mix as an internalcounting control. The percent (%) of cell killing may be calculatedbased on non-TDB treated controls. Activated T cells may be detected byCD69 and CD25 surface expression using anti-CD69-FITC and anti-CD25-PE.

D. Immunoconjugates

The invention also provides immunoconjugates comprising an anti-FcRH5antibody, for example an anti-FcRH5 multispecific antibody, for examplean FcRH5 TDB described herein conjugated to one or more cytotoxicagents, such as chemotherapeutic agents or drugs, growth inhibitoryagents, toxins (e.g., protein toxins, enzymatically active toxins ofbacterial, fungal, plant, or animal origin, or fragments thereof), orradioactive isotopes.

In one embodiment, an immunoconjugate is an antibody-drug conjugate(ADC) in which an antibody is conjugated to one or more drugs, includingbut not limited to a maytansinoid (see U.S. Pat. Nos. 5,208,020,5,416,064 and European Patent EP 0 425 235 B1); an auristatin such asmonomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S.Pat. Nos. 5,635,483 and 5,780,588, and 7,498,298); a dolastatin; acalicheamicin or derivative thereof (see U.S. Pat. Nos. 5,712,374,5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and5,877,296; Hinman et al. Cancer Res. 53:3336-3342, 1993; and Lode et al.Cancer Res. 58:2925-2928, 1998); an anthracycline such as daunomycin ordoxorubicin (see Kratz et al. Current Med. Chem. 13:477-523, 2006;Jeffrey et al. Bioorganic & Med. Chem. Letters 16:358-362, 2006; Torgovet al. Bioconj. Chem. 16:717-721, 2005; Nagy et al. Proc. Natl. Acad.Sci. USA 97:829-834, 2000; Dubowchik et al. Bioorg. & Med. Chem. Letters12:1529-1532, 2002; King et al. J. Med. Chem. 45:4336-4343, 2002; andU.S. Pat. No. 6,630,579); methotrexate; vindesine; a taxane such asdocetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; atrichothecene; and CC1065.

In another embodiment, an immunoconjugate comprises an anti-FcRH5antibody (for example, an anti-FcRH5 multispecific antibody, for examplean FcRH5 TDB) described herein conjugated to an enzymatically activetoxin or fragment thereof, including but not limited to diphtheria Achain, nonbinding active fragments of diphtheria toxin, exotoxin A chain(from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin Achain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins,Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordicacharantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor,gelonin, mitogellin, restrictocin, phenomycin, enomycin, and thetricothecenes.

In another embodiment, an immunoconjugate comprises an anti-FcRH5antibody (for example, an anti-FcRH5 multispecific antibody, for examplean FcRH5 TDB) described herein conjugated to a radioactive atom to forma radioconjugate. A variety of radioactive isotopes are available forthe production of radioconjugates. Examples include At²¹¹, I¹³¹, I¹²⁵,Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes ofLu. When the radioconjugate is used for detection, it may comprise aradioactive atom for scintigraphic studies, for example tc99m or I123,or a spin label for nuclear magnetic resonance (NMR) imaging (also knownas magnetic resonance imaging, mri), such as iodine-123 μgain,iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-111,gadolinium, manganese or iron.

Conjugates of an antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al. Science 238:1098, 1987.Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of a cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al. Cancer Res. 52:127-131, 1992;U.S. Pat. No. 5,208,020) may be used.

The immunuoconjugates or ADCs herein expressly contemplate, but are notlimited to such conjugates prepared with cross-linker reagentsincluding, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS,MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS,sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate) which are commercially available(e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A).

E. Pharmaceutical Formulations

Pharmaceutical formulations of an anti-FcRH5 antibody of the invention(e.g., a bispecific anti-FcRH5 antibody of the invention that binds toFcRH5 and a second biological molecule, e.g., CD3, e.g., an FcRH5 TDB)are prepared by mixing such antibody having the desired degree of puritywith one or more optional pharmaceutically acceptable carriers, buffers,stabilizers, and/or preservatives (Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980)), in the form of lyophilizedformulations or aqueous solutions. Pharmaceutically acceptable carriersare generally nontoxic to recipients at the dosages and concentrationsemployed, and include, but are not limited to: buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as polyethylene glycol (PEG). Exemplarypharmaceutically acceptable carriers herein further includeinsterstitial drug dispersion agents such as soluble neutral-activehyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, BaxterInternational, Inc.). Certain exemplary sHASEGPs and methods of use,including rHuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined withone or more additional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody formulations are described in U.S. Pat.No. 6,267,958. Aqueous antibody formulations include those described inU.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulationsincluding a histidine-acetate buffer.

The formulation herein may also contain more than one active ingredientsas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. For example, it may be desirable to further provide an additionaltherapeutic agent (e.g., a chemotherapeutic agent, a cytotoxic agent, agrowth inhibitory agent, and/or an anti-hormonal agent, such as thoserecited herein above). Such active ingredients are suitably present incombination in amounts that are effective for the purpose intended.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

In some instances, the composition may comprise a PD-1 axis bindingantagonist and/or an additional therapeutic agent. For example, the PD-1axis binding antagonist may be selected from the consisiting ofMPDL3280A (atezolizumab), YW243.55.S70, MDX-1105, MED1473 (durvalumab),and MSB0010718C (avelumab), MDX 1106 (nivolumab), MK-3475(pembrolizumab), CT-011 (pidilizumab), MEDI-0680 (AMP-514), PDR001,REGN2810, and BGB-108. Additionally, a composition comprising ananti-FcRH5 antibody of the invention (e.g., an FcRH5 TDB) may comprise asteroid, an immunomodulator (IMiD), a proteosome inhibitor (PI), or acombination thereof.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, for example, films, or microcapsules.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

F. Therapeutic Methods and Compositions

Any of the anti-FcRH5 antibodies of the invention (e.g., bispecificanti-FcRH5 antibodies of the invention that bind to FcRH5 and a secondbiological molecule, e.g., CD3, e.g., an FcRH5 TDB) may be used intherapeutic methods.

In one aspect, an anti-FcRH5 antibody for use as a medicament isprovided. In further aspects, an anti-FcRH5 antibody for use in treatingor delaying progression of a cell proliferative disorder (e.g., cancer,e.g., an FcRH5-positive cancer, e.g., multiple myeloma (MM)) and/orenhancing an immune function in an individual is provided. In certainembodiments, an anti-FcRH5 antibody for use in a method of treatment isprovided. In certain embodiments, the invention provides an anti-FcRH5antibody for use in a method of treating an individual having a cellproliferative disorder (e.g., cancer, e.g., an FcRH5-positive cancer,e.g., MM)) comprising administering to the individual an effectiveamount of the anti-FcRH5 antibody. In one such embodiment, the methodfurther comprises administering to the individual an effective amount ofat least one additional therapeutic agent, for example, as describedbelow.

In further embodiments, the invention provides an anti-FcRH5 antibodyfor use in enhancing immune function in an individual having a cellproliferative disorder, such as an FcRH5-positive cancer (e.g., MM). Incertain embodiments, the invention provides an anti-FcRH5 antibody foruse in a method of enhancing immune function in an individual having acell proliferative disorder comprising administering to the individualan effective of the anti-FcRH5 antibody to activate effector cells(e.g., T cells, e.g., CD8+ and/or CD4+ T cells) capable of exerting acytotoxic and/or an apoptotic effect on target cells (e.g., a cellexpressing a second biological molecule recognized by an anti-FcRH5antibody of the invention, such as a FcRH5 TDB antibody of theinvention) in an individual. The anti-FcRH5 antibody may bind to both anFcRH5 molecule located on a target cell and a CD3 molecule located on animmune effector cell. The target cell may be plasma cell, such as a longor short lived plasma cell. Additionally, the target cell may be amyeloma cell. An “individual” according to any of the above embodimentsmay be a human.

In a further aspect, the invention provides for the use of an anti-FcRH5antibody in the manufacture or preparation of a medicament. In oneembodiment, the medicament is for treatment of a cell proliferativedisorder (e.g., cancer, e.g., an FcRH5-positive cancer, e.g., MM)). In afurther embodiment, the medicament is for use in a method of treating acell proliferative disorder comprising administering to an individualhaving a cell proliferative disorder or an effective amount of themedicament. In one such embodiment, the method further comprisesadministering to the individual an effective amount of at least oneadditional therapeutic agent, for example, as described below.

In a further embodiment, the medicament is for activating effector cellsactivate effector cells (e.g., T cells, e.g., CD8+ and/or CD4+ T cells)capable of exerting a cytotoxic and/or an apoptotic effect on targetcells (e.g., a cell expressing a second biological molecule recognizedby an anti-FcRH5 antibody of the invention, such as a FcRH5 TDB antibodyof the invention) in an individual. In a further embodiment, themedicament is for use in a method of enhancing immune function in anindividual having a cell proliferative disorder comprising administeringto the individual an amount effective of the medicament to activateeffector cells (e.g., T cells, e.g., CD8+ and/or CD4+ T cells), expand(increase) an effector cell population, reduce a target cell (e.g., acell expressing a second biological molecule recognized by an anti-FcRH5antibody of the invention, such as a FcRH5 TDB antibody of theinvention) population, and/or kill a target cell (e.g., target tumorcell). An “individual” according to any of the above embodiments may bea human.

In a further aspect, the invention provides a method for treating a cellproliferative disorder (e.g., cancer). The FcRH5-positive cancer may bea B cell cancer. In some instances, the B-cell cancer may be multiplemyeloma (MM), chronic lymphoid leukemia (CLL), mantle cell lymphoma(MCL), diffuse large B-cell lymphoma (DLBCL), and/or follicular lymphoma(FL). In particular instances, the B cell cancer is MM. In otherinstances, the FcRH5-positive cancer is a B cell cancer. In oneembodiment, the method comprises administering to an individual havingsuch a cell proliferative disorder (e.g., cancer, e.g., FcRH5-positivecancer, e.g., multiple myeloma (MM)) an effective amount of ananti-FcRH5 antibody. In one such embodiment, the method furthercomprises administering to the individual an effective amount of atleast one additional therapeutic agent, for example, as described below.An “individual” according to any of the above embodiments may be ahuman.

Antibodies of the invention can be used either alone or in combinationwith other agents in a therapy. For instance, an antibody of theinvention may be co-administered with at least one additionaltherapeutic agent. In certain embodiments, an additional therapeuticagent is a PD-1 axis binding antagonist such as MPDL3280A(atezolizumab), YW243.55.S70, MDX-1105, MED1473 (durvalumab), andMSB0010718C (avelumab), MDX 1106 (nivolumab), MK-3475 (pembrolizumab),CT-011 (pidilizumab), MEDI-0680 (AMP-514), PDR001, REGN2810, and/orBGB-108. Additionally, a steroid, an immunomodulator (IMiD), aproteosome inhibitor (PI), or a combination thereof may also beadministered to a subject. In one embodiment, the glucocorticoid isdexamethasone. In some embodiments, the IMiD is lenalidomide. In someembodiments, the PI is bortezomib.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate formulations), and separate administration, in which case,administration of the antibody of the invention can occur prior to,simultaneously, and/or following, administration of the additionaltherapeutic agent or agents. In one embodiment, administration of theanti-FcRH5 antibody and administration of an additional therapeuticagent occur within about one month, or within about one, two or threeweeks, or within about one, two, three, four, five, or six days, of eachother.

An antibody of the invention (and/or any additional therapeutic agent)can be administered by any suitable means, including intravenously,subcutaneously, intramuscularly, topically, orally, transdermally,intraperitoneally, intraorbitally, by implantation, by inhalation,intrathecally, intraventricularly, or intranasally. Dosing can be by anysuitable route, for example, by injections, such as intravenous orsubcutaneous injections, depending in part on whether the administrationis brief or chronic. Various dosing schedules including but not limitedto single or multiple administrations over various time-points, bolusadministration, and pulse infusion are contemplated herein.

Antibodies of the invention would be formulated, dosed, and administeredin a fashion consistent with good medical practice. Factors forconsideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Theantibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question.

The effective amount of such other agents depends on the amount ofantibody present in the formulation, the type of disorder or treatment,and other factors discussed above. These are generally used in the samedosages and with administration routes as described herein, or aboutfrom 1 to 99% of the dosages described herein, or in any dosage and byany route that is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease (e.g., a proliferative celldisorder, e.g., cancer, e.g., a FcRH5-positive cancer, e.g., MM)), theappropriate dosage of an antibody of the invention (when used alone orin combination with one or more other additional therapeutic agents)will depend on the type of disease to be treated, the type of antibody,the severity and course of the disease, whether the antibody isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the antibody, and thediscretion of the attending physician. The antibody is suitablyadministered to the patient at one time or over a series of treatments.

As a general proposition, the therapeutically effective amount of theanti-FcRH5 antibody administered to a human will be in the range ofabout 0.01 to about 100 mg/kg of patient body weight whether by one ormore administrations. In some embodiments, the antibody used is about0.01 to about 0.01 to about 55 mg/kg, 50 mg/kg, 0.01 to about 45 mg/kg,about 0.01 to about 40 mg/kg, about 0.01 to about 35 mg/kg, about 0.01to about 30 mg/kg, about 0.01 to about 25 mg/kg, about 0.01 to about 20mg/kg, about 0.01 to about 15 mg/kg, about 0.01 to about 10 mg/kg, about0.01 to about 5 mg/kg, or about 0.01 to about 1 mg/kg administereddaily, for example. In one embodiment, an anti-FcRH5 antibody describedherein is administered to a human at a dose of about 100 mg, about 200mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200mg, about 1300 mg or about 1400 mg on day 1 of 21-day cycles. The dosemay be administered as a single dose or as multiple doses (e.g., 2 or 3doses), such as infusions. For repeated administrations over severaldays or longer, depending on the condition, the treatment wouldgenerally be sustained until a desired suppression of disease symptomsoccurs. One exemplary dosage of the antibody would be in the range fromabout 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5mg/kg, 2.0 mg/kg, 4.0 mg/kg, or 10 mg/kg (or any combination thereof)may be administered to the patient. In some embodiments, the anti-FcRH5antibody is administered at a dosage of about 0.01 mg/kg/wk to about 10mg/kg/wk. In other embodiments, the anti-FcRH5 antibody is administeredat a dosage of 0.1 mg/kg/wk to about 10 mg/kg/wk. In other embodiments,anti-FcRH5 antibody is administered at a dosage of about 1 mg/kg/wk.

Such doses may be administered intermittently, for example, every weekor every three weeks (e.g., such that the patient receives from abouttwo to about twenty, or, for example, about six doses of the anti-FcRH5antibody). An initial higher loading dose, followed by one or more lowerdoses may be administered. The progress of this therapy is easilymonitored by conventional techniques and assays.

In some embodiments, the methods may further comprise an additionaltherapy. The additional therapy may be radiation therapy, surgery,chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy,immunotherapy, bone marrow transplantation, nanotherapy, monoclonalantibody therapy, or a combination of the foregoing. The additionaltherapy may be in the form of adjuvant or neoadjuvant therapy. In someembodiments, the additional therapy is the administration of smallmolecule enzymatic inhibitor or anti-metastatic agent. In someembodiments, the additional therapy is the administration of side-effectlimiting agents (e.g., agents intended to lessen the occurrence and/orseverity of side effects of treatment, such as anti-nausea agents,etc.). In some embodiments, the additional therapy is radiation therapy.In some embodiments, the additional therapy is surgery. In someembodiments, the additional therapy is a combination of radiationtherapy and surgery. In some embodiments, the additional therapy isgamma irradiation. In some embodiments, the additional therapy may be aseparate administration of one or more of the therapeutic agentsdescribed above.

G. Methods and Compositions for Diagnostics and Detection

In certain embodiments, any of the anti-FcRH5 antibodies of theinvention is useful for detecting the presence of FcRH5 in a biologicalsample. The term “detecting” as used herein encompasses quantitative orqualitative detection. In certain embodiments, a biological samplecomprises a cell or tissue.

In one embodiment, an anti-FcRH5 antibody for use in a method ofdiagnosis or detection is provided. In a further aspect, a method ofdetecting the presence of naturally occurring FcRH5 in a biologicalsample is provided. In certain embodiments, the method comprisescontacting the biological sample with an anti-FcRH5 antibody asdescribed herein under conditions permissive for binding of theanti-FcRH5 antibody to the naturally occurring FcRH5, and detectingwhether a complex is formed between the anti-FcRH5 antibody andnaturally occurring FcRH5. Such method may be an in vitro or in vivomethod. In some instances, the biological sample is a blood sample. Insome instances, the subject is a human.

In certain embodiments, labeled anti-FcRH5 antibodies are provided.Labels include, but are not limited to, labels or moieties that aredetected directly (such as fluorescent, chromophoric, electron-dense,chemiluminescent, and radioactive labels), as well as moieties, such asenzymes or ligands, that are detected indirectly, e.g., through anenzymatic reaction or molecular interaction. Exemplary labels include,but are not limited to, the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and¹³¹I,fluorophores such as rare earth chelates or fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone,luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S.Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,horseradish peroxidase (HRP), alkaline phosphatase, 3-galactosidase,glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase,galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclicoxidases such as uricase and xanthine oxidase, coupled with an enzymethat employs hydrogen peroxide to oxidize a dye precursor such as HRP,lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,bacteriophage labels, stable free radicals, and the like.

H. Articles of Manufacture

In another aspect, the invention features an article of manufacturecontaining materials useful for the treatment, prevention, and/ordiagnosis of proliferative cell disorders (e.g., cancer, e.g., anFcRH5-positive cancer, e.g., multiple myeloma (MM)) in a subject (e.g.,a human). The article of manufacture comprises a container and a labelor package insert on or associated with the container. Suitablecontainers include, for example, bottles, vials, syringes, IV solutionbags, etc. The containers may be formed from a variety of materials suchas glass or plastic. The container holds a composition which is byitself or combined with another composition effective for treating,preventing, and/or diagnosing the condition and may have a sterileaccess port (for example the container may be an intravenous solutionbag or a vial having a stopper pierceable by a hypodermic injectionneedle). At least one active agent in the composition is an antibody ofthe invention. The label or package insert indicates that thecomposition is used for treating the condition of choice. Moreover, thearticle of manufacture may comprise (a) a first container with acomposition contained therein, wherein the composition comprises anantibody of the invention; and (b) a second container with a compositioncontained therein, wherein the composition comprises a further cytotoxicor otherwise therapeutic agent. The article of manufacture in thisembodiment of the invention may further comprise a package insertindicating that the compositions can be used to treat a particularcondition. Alternatively, or additionally, the article of manufacturemay further comprise a second (or third) container comprising apharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution, anddextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, and syringes. In some instances, the kit includes apackage insert comprising instructions for using the antibody fortreating or delaying progression of an FcRH5-positive cancer (e.g., MM)in a subject. In other instances, the kit includes a package insertcomprising instructions for using the antibody for enhancing immunefunction in a subject having an FcRH5-positive cancer (e.g., MM).

III. EXAMPLES

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Example 1. Generation of Anti-FcRH5 Antibodies Immunizations andScreening

BALB/c mice (Charles River, Hollister, Calif.) were immunized with 2 μg,10 μg, or 100 μg/injection per mouse. Antigens were suspended inmonophosphoryl lipid A (MPL)/trehalose dicorynomycolate (TDM) (Ribi)adjuvant or Freund's adjuvant and injected into the footpad, peritoneum,or the base of the tail of each immunized animal. Mice received a totalof 9 to 18 doses and 1 to 2 prefusion boosts in PBS alone via footpad,intraperitoneally (IP), and/or hock routes 2 to 4 days prior to fusion.Immunization Campaign A, described in U.S. Pub. No. 2015/0098900,produced the parental anti-FcRH5 antibody 1G7, which is optimized andfurther characterized herein.

A. Immunization Campaign B

To produce isoform-specific antibodies for the membrane-proximalIg-domain of FcRH5, mice were immunized with E11 protein (amino acids745-850 of SEQ ID NO: 114) with an N-terminal His-expression tag thatwas expressed in CHO cells. Five mice were immunized with 10 μg ofHis-tagged E11 protein in Ribi adjuvant via the IP route. Mice received18 doses followed by two prefusion boosts in PBS alone via the IP routeseven and four days prior to fusion. After 18 doses of the recombinantE11 protein, serum was analyzed for FcRH5-binding antibodies using FACSand by titration in ELISA using E11 as the antigen. Significantreactivity was detected to SVT2 cells that expressed full-length humanFcRH5, full-length cyno FcRH5, or E11 protein, but not vectortransfected SVT2 cells, indicating that FcRH5 binding antibodies werepresent in the sera of all five immunized mice. After 20 doses,lymphocytes from the immunized mice were electrofused withP3X63Ag8U.1.22 mouse myeloma cells.

Clones were tested for binding to recombinant human and cyno E11proteins by ELISA. Eight clones that were cross-reactive to both humanand cyno FcRH5 in ELISA were tested for binding to SVT2 cells thatexpressed full-length human FcRH5, full-length cyno FcRH5, or E11protein by FACS. All eight clones bound SVT2 cells expressing the humanE11 domain, transmembrane domain, and cytoplasmic domains.

To test binding to cancer cells which endogenously express FcRH5 orwhich were transfected with FcRH5, the cells were lifted using EDTA/PBS,and 1×10⁵ cells were suspended in 100 μl and incubated with primaryantibodies (1 volume of non-lgG quantitated subclone supernatant, 4μg/ml IgG quantified subclone supernatant or 2 μg/ul purified monoclonalantibodies). Cells were washed twice with FACS buffer (PBS, 1% BSA, 2 mMEDTA) and incubated with 1:1000 dilution of goat anti-mouse secondarylabeled with PE or 1:100 of goat anti-mouse APC. Cells were washed twicewith FACS buffer and flow cytometry analysis was done on a FACSCalibur.Direct Xenon-labeling of antibodies was carried out according tomanufacturer's protocol (Invitrogen), when indicated. However, onlythree of the clones bound to cells that expressed full-length humanFcRH5 and none bound to cells that expressed full-length cyno FcRH5(Table 2).

TABLE 2 Binding of mAbs from Immunization Campaign B in FACS to cellsoverexpressing human FcRH1, 2, 3, 4, and 5, human E11 domain, andcynomolgus FcRH5 Binding to FACS to SVT2 cells expressing: Human HumanHuman Human Human Human E11- Cynomolgus mAb FcRH1 FcRH2 FcRH3 FcRH4FcRH5 TM-ICD* FcRH5 1E6 − − − − + + − 3D8 − − − − − + − 8G8 − − − − − +− 11A11 − − − − − + − 10D9 − − − − + + − 11H6 − − − − − + − 13D2 − − −− + + − 14E9 − − − − − + − *E11 domain, transmembrane domain, andcytoplasmic domains of human FcRH5

B. Immunization and mAb Characterization Campaign C For ImmunizationCampaign C, N-terminally His-tagged E11 protein was produced in CHOcells.

Mice were immunized with an initial injection of 100 μg of E11 proteinin complete Freund's adjuvant in the base of the tail. Mice received atotal of 17 doses, followed by one prefusion boost in PBS alone via theIP and hock routes two days prior to fusion (Table 3).

TABLE 3 Immunization schedule for the Immunization Campaign C InjectionVolume Volume Protein number Adjuvant Route per site per animal amountAntigen 1 CFA Base of tail 100 μl 100 μl 100 μg human E11 2 IFAIntraperitoneal 100 μl 100 μl 50 μg human E11 IFA Hock  50 μl 100 μl 50μg human E11 3 IFA Base of tail 100 μl 100 μl 50 μg human E11 IFASubcutaneous 100 μl 100 μl 50 μg human E11 IFA Intraperitoneal 100 μl100 μl 50 μg human E11 4 IFA Hock 100 μl 100 μl 50 μg human E11 IFAIntraperitoneal 100 μl 100 μl 50 μg human E11 5 IFA Intraperitoneal 100μl 100 μl 50 μg human E11 IFA Subcutaneous 100 μl 100 μl 50 μg human E11IFA Base of tail 100 μl 100 μl 50 μg human E11 6 IFA Intraperitoneal 100μl 100 μl 50 μg human E11 IFA Hock 100 μl 100 μl 50 μg human E11 7 IFABase of tail 100 μl 100 μl 50 μg human E11 IFA Subcutaneous 100 μl 100μl 50 μg human E11 IFA Intraperitoneal 100 μl 100 μl 50 μg human E11 8Sterile PBS Intraperitoneal 100 μl 100 μl 50 μg human E11 9 Sterile PBSIntraperitoneal 100 μl 100 μl 50 μg human E11 10 Ribi Intraperitoneal100 μl 100 μl 10 μg human E11 11 Ribi Intraperitoneal 100 μl 100 μl 10μg human E11 12 Ribi Intraperitoneal 100 μl 100 μl 10 μg cynomolgus E11and human E11 13 Ribi Intraperitoneal 100 μl 100 μl 10 μg cynomolgus E11and human E11 14 Ribi Intraperitoneal 100 μl 100 μl 3.3 μg cynomolgusE11 and human E11 Ribi Hock 100 μl 100 μl 3.3 μg cynomolgus E11 andhuman E11 15 Ribi Base of tail 100 μl 100 μl 3.3 μg cynomolgus E11 andhuman E11 Ribi Subcutaneous 100 μl 100 μl 3.3 μg cynomolgus E11 andhuman E11 Ribi Intraperitoneal 100 μl 100 μl 3.3 μg cynomolgus E11 andhuman E11 16 Ribi Intraperitoneal 100 μl 100 μl 3.3 μg cynomolgus E11and human E11 Ribi Hock 100 μl 200 μl 6.6 μg cynomolgus E11 and humanE11 17 Ribi Base of tail 100 μl 100 μl 3.3 μg cynomolgus E11 and humanE11 Ribi Subcutaneous 100 μl 100 μl 3.3 μg cynomolgus E11 and human E11Ribi Intraperitoneal 100 μl 100 μl 3.3 μg cynomolgus E11 and human E11Pre-fusion Sterile PBS Intraperitoneal 100 μl 100 μl 3.3 μg cynomolgusE11 and human E11 boost Sterile PBS Hock 100 μl 200 μl 6.6 μg cynomolgusE11 and human E11

After 15 doses of the recombinant E11 protein followed by co-injectionsof the recombinant human and cyno E11 proteins, serum was analyzed forFcRH5-binding antibodies using FACS and by titration in ELISA using E11protein as the antigen. Significant reactivity was detected to SVT2cells that expressed full-length human FcRH5, full-length cyno FcRH5, orthe human E11 domain, transmembrane domain, and cytoplasmic domains, butnot vector transfected SVT2 cells, indicating that FcRH5 bindingantibodies were present in the sera of all five immunized mice. Afterthe final boost in PBS, lymphocytes from the immunized mice wereelectrofused with P3X63Ag8U.1.22 mouse myeloma cells.

Clones were tested for binding to recombinant human and cyno E11proteins and binding to SVT2 cells that expressed full-length humanFcRH5; full-length cyno FcRH5; full-length human FcRH1, FcRH2, FcRH3, orFcRH4; or human E11 domain, transmembrane domain, and cytoplasmicdomains of FcRH5 by FACS. There were 44 human E11 protein-positiveclones and 32 cyno E11 protein-positive clones in ELISA. Out of those, atotal of 16 clones were identified that bound to cells that expressedhuman FcRH5 and cells that expressed cyno FcRH5, but not cells thatexpressed FcRH1, FcRH2, FcRH3, or FcRH4, indicative of specific FcRH5cross-species reactivity (Table 4).

TABLE 4 Binding properties of monoclonal antibodies obtained fromImmunization Campaign C Binding in FACS to SVT2 cells expressing: MOLP-2Human Human Human Human Human Human E11- Cynomolgus FACS KD human KDcynomolgus mAb FcRH1 FcRH2 FcRH3 FcRH4 FcRH5 TM-ICD* FcRH5 binding E11domain§ E11 domain§ 15G8 − − − − + + +/− +/− 0.5 nM 9.1 nM 24H5 − − −− + + + + 3.2 nM 255 nM 16H10 − − − − + + + + 4.6 nM 338 nM 10A7 − − −− + + + + 4.9 nM 229 nM 3C5 − − − − + + + + 45 nM 81 nM 17B1 − − −− + + + + 68 nM 27 nM 23E2 − − − − + + + +  Not done¶  Not done¶ 9B3 − −− − + + + − Not done Not done 23B8 − − − − + + + − Not done Not done23G3 − − − − + + + − Not done Not done 24F5 − − − − + + + − Not done Notdone 24G6 − − − − + + + − Not done Not done 24C4 − − − − +/− + +/− − Notdone Not done 3C2 − − − − + + +/− − Not done Not done 6H3 − − − − +/− ++/− − Not done Not done 1D10 − − + − + + + Not done Not done Not done12B6 + + + − + + + Not done Not done Not done 16A6 + + + − + + + Notdone Not done Not done 11C9 − − − − + + − Not done Not done Not done11H10 − − − − + + − Not done Not done Not done 12E10 − − − − + + − Notdone Not done Not done 16BS − − − − + + − Not done Not done Not done16H1 − − − − + + − Not done Not done Not done 17A6 − − − − + + − Notdone Not done Not done 2A10 − − − − + + − Not done Not done Not done 2F9− − − − + + − Not done Not done Not done 3E3 − − − − + + − Not done Notdone Not done 5G9 − − − − + + − Not done Not done Not done 6G4 − − − − +− − Not done Not done Not done 7E2 − − − − +/− + − Not done Not done Notdone 8E2 − − − − + + − Not done Not done Not done 9A6 − − − − + + − Notdone Not done Not done 13C8 − − − − − + − Not done Not done Not done16E5 − − − − − + − Not done Not done Not done 22C9 − − − − − + − Notdone Not done Not done 23B9 − − − − − + − Not done Not done Not done 4H3− − − − − + − Not done Not done Not done 5B1 − − − − − + − Not done Notdone Not done 1B9 − − − − − +/− − Not done Not done Not done 2C3 − − − −− +/− − Not done Not done Not done 3G2 − − − − − +/− − Not done Not doneNot done 2G4 − − − − − − − Not done Not done Not done *E11 domain;transmembrane domain, and cytoplasmic domains of human FcRH5. §Surfaceplasmon resonance BIACORE ® KD, monovalent affinity. Human E11 domainexperiments were performed 1 to 3 times and averaged. Cynomolgus E11domain experiments were performed once for each antibody. ¶Norecombinant clones were obtained for antibody 23E2.

Purified IgG from the 16 selected monoclonal antibody (mAb) clones werefurther characterized for binding to cells that express human FcRH5endogenously (MOLP-2 cells). A total of eight mAb clones were shown tobind MOLP-2 myeloma cells. Two of these eight mAb clones were found tobe redundant based on sequence analysis and one was not molecularlycloned. The six molecularly cloned MOLP-2-positive mAb clones wereexpressed recombinantly as murine IgG2a and tested for affinity bysurface plasmon resonance in a BIACORE® T200 apparatus in an IgG captureformat.

Briefly, test antibodies were captured on flow cells 2, 3 or 4 with flowcell 1 as a reference on a protein A Series S CM5 chip (GE LifeSciences, Piscataway, N.J.). An FcRH5 protein fragment was used asanalyte, with a flow rate of 30 μl/minute. Between injections thecapture surface was regenerated by a 30 second injection of 10 mMglycine, pH 1.5 at a flow rate of 10 μl/minute. Interactions wereassessed at 25° C. in 10 mM HEPES pH 7.4, 150 mM NaCl, 0.05% Tween 20(HBSP). Reference data from the reference flow cell and from injectionof buffer alone was subtracted prior to kinetic analysis. Kineticinformation was calculated by fitting data to a 1:1 binding model.Reference subtraction and data fitting were performed usingBIAevaluation software (GE Life Sciences, Piscataway, N.J.).

The monovalent affinities of these antibodies for the human E11 domainranged from 0.5 nM to 68 nM while the affinities for cynomolgus E11domain ranged from 9.1 nM to 338 nM (Table 4). Only one clone had anaffinity for human E11 domain that was higher than antibody 1G7, mAb15G8. However, the K_(D) difference between human and cynomolgus E11domains was about 20-fold, which rendered this clone unsuitable forfurther clinical development. In addition, despite the high affinity ofthis antibody clone for the isolated human domain E11, the binding ofthis antibody clone to MOLP-2 cells was very weak; it was weaker thanthe clones with lower affinities for isolated human E11 domain,indicating that this clone did not bind well to the native human FcRH5protein.

Example 2. Generation and Characterization of Anti-FcRH5 AntibodyVariants from Immunization Campaigns A and C

A. Humanization of Anti-FcRH5 Monoclonal Antibodies from ImmunizationCampaign A

Anti-FcRH5 antibody 1G7 from Immunization Campaign A, described in U.S.Pub. No. 2015/0098900, was humanized by the HVR graft method aspreviously described (Presta et al. Cancer Res. 57:4593-4599, 1997),except that consensus VH4 and Vκ1 frameworks (Dennis, Current Trends inMonoclonal Ant. Develop. and Manufac. 9-28, 2010) were used as acceptorframeworks. The heavy chain graft also included murine residues atframework Kabat positions 37, 48, 67, 71, 73, 78, 93, and 94, and thelight chain graft also included murine residues at framework positions36 and 43 for proper HVR presentation and VH/VL domain contact. Residuenumbers are according to Kabat et al. (Sequences of proteins ofimmunological interest, 5th Ed., Public Health Service, NationalInstitutes of Health, Bethesda, Md., 1991). The sequence of humanized1G7, herein referred to as hu1G7.v1 or 1G7.v1, is shown in FIGS. 2 and3.

B. Affinity Maturation and Characterization of Hu 1G7.v1 Variants

To enhance potency, humanized antibody hu1G7.v1 was affinity maturedusing phage display. Briefly, randomized libraries of the Fab antibodyfragment on the surface of M13 bacteriophage were displayed and pannedfor binders to biotinylated FcRH5 protein fragment. Favored mutationswere identified by DNA sequencing of individual clones. These favoredmutations were postulated to lead to variants with improved affinity (Liet al. MAbs. 6:437-445, 2014). Antibody clones with selected mutationswere tested for affinity by surface plasmon resonance (FIGS. 42 and 43).

Briefly, a human IgG capture surface was generated on a Series S CM5chip using amine coupling and the human IgG capture kit (GE LifeSciences, Piscataway, N.J.). Test antibodies were then captured on flowcells 2, 3, or 4, with flow cell 1 used as a reference. An FcRH5 proteinfragment was used as the analyte, with a flow rate of >30 μl/minute.Between injections the capture surface was regenerated by a 30-secondinjection of 3M magnesium chloride or 10 mM glycine, pH 1.5.Interactions were assessed at 25° C. in 10 mM HEPES pH7.4, 150 mM NaCl,and 0.05% Tween 20 (HBSP). Reference data from the reference flow celland from injection of buffer alone were subtracted prior to kineticanalysis. Kinetic information was calculated by fitting data to a 1:1binding model. Reference subtraction and data fitting were performedusing BIAevaluation software (GE Life Sciences, Piscataway, N.J.).

For initial affinity screening experiments, antibodies were captureddirectly from clarified culture media collected following transienttransfection of Expi293 cells (Invitrogen) (FIGS. 42 and 43). Theability of each antibody to bind an FcRH5 protein fragment was assessedat 100 nM and 500 nM (Expt. 1, FIGS. 42 and 43) or 0 nM, 20 nM, 100 nM,and 500 nM (Expt. 2, FIGS. 42 and 43). Association and dissociation weremonitored at a flow rate of 30 μl/min, for 180 s and 600 s,respectively. All sequences present within FIG. 42 are disclosed withinSEQ ID NOs: 4-6 (i.e., the FcRH5 L1, L2, L3 broad consensus). Allsequences present within FIG. 43 are disclosed within SEQ ID NOs: 1-3(i.e., the FcRH5 H1, H2, H3 broad consensus). Affinity improvements ofup to 5.2-fold were observed (FIG. 42). As each individual mutation didnot provide the improvements sought, it was decided to combine variousmutations with a focus on VL mutations S30R, I32L, A51G, S52Y, Y53N,T56S, H91Q, Y92F, S93Q, and S94P and VH mutations S28K, L29T, and S56T.

Antibody phage display libraries were designed using both hard and softsite-directed mutagenesis. Libraries L188 and L189 used soft mutagenesisto mutate VL residues 27-34, 50-56, 91-94, and 96 (L188) or VH residues28-35, 50-58, and 95-100d (L189). At mutagenesis positions, softmutagenesis oligonucleotides utilized 91% parent-encoding nucleotide and3% of each other nucleotide. Libraries L190 and L191 used hardmutagenesis (NNKcodons) to mutate VL residues 27-34, 50-56, and 89-96(L190) or VH residues 28-35, 50-58 and 95-100d (L191), one codon at atime, with up to three HVRs mutated simultaneously (Li et al. MAbs.6:437-445, 2014).

Up to four rounds of selection for binding to FcRH5 protein fragmentwere performed. Selections were performed in both solid phase (targetprotein adsorbed directly onto microplates) and in solution. Forsolution selections, biotinylated target protein was used to enablecapture of phage-antibody-target complexes. Clones with selectedmutations were expressed in Expi293 cells (Thermo Fisher Scientific,Waltham, Mass.), and the supernatants screened by surface plasmonresonance.

Antibody phage display libraries were generated by Kunkel mutagenesis(Kunkel et al. Methods in Enzymology, 154:367-382, 1987) using astemplate single-stranded DNA produced in CJ236 bacteria and purifiedusing the Qiagen QIAprep Spin M13 Kit (QIAGEN, Inc., Valencia, Calif.).Template DNA for initial affinity maturation libraries contained stopcodons in those HVRs selected for mutagenesis; template DNA for thesubsequent “focused” phage library described below did not utilizetemplate HVR stop codons. Unless stated otherwise, residues in antibodyvariable regions are numbered according to the Kabat system. Enzymeswere purchased from New England Biolabs (Ipswich, Mass.). Kunkelmutagenesis reactions were cleaned up using QIAprep mini spin columns(QIAGEN, Inc., Valencia, Calif.) and transformed into XLI Blue E. coli(Agilent Technologies, Santa Clara, Calif.) by electroporation.Following recovery in SOC medium at 37° C., cultures were infected withhelper phage and incubated at 37° C. for an additional 0.5-2 hoursbefore addition of antibiotics carbenicillin and kanamycin. Cultureswere then incubated at 37° C. for an additional 4-5 hours followed byfurther incubation overnight at 30° C. Bacteriophage were purified fromclarified culture medium by precipitation with approximately 1/6 volumeof precipitation reagent (20% PEG, 2.5M sodium chloride). Precipitatedphage were pelleted by centrifugation and solubilized in phosphatebuffered saline (PBS). The solution was further clarified bycentrifugation at 14,000 g. Purified phage were stored in 50% glycerolin a −20° C. freezer and re-solubilized in PBS before use.

In a rational design approach, six VL mutations identified from thephage experiments (S30R, I32L, A51G, T56S, H91Q, and S94P) were combinedwith additional VL humanization mutation S43A and evaluated in pairs inorder to assess compatibility (FIG. 44). For compatibility assessment ofselected VL mutations (FIG. 44), antibodies were captured directly fromclarified culture media collected following transient transfection ofExpi293 cells. Binding of FcRH5 protein fragment was assessed at 6 nM,19 nM, 56 nM, 167 nM, and 500 nM at a flow rate of 30 μl/min.Association and dissociation were monitored for 300 s and 600 s,respectively. The sequences disclosed in FIG. 44 are individual aminoacids (i.e., the number at the top of the column is the amino acidnumber within the antibody sequence). The results indicated that the VLmutations I32L and H91Q, while individually improving affinity, did notbenefit affinity when combined. However, the other sequences tested gaveincremental affinity improvements of up to four-fold compared to thehu1G7.v1 control. Based on these results, antibodies hu1G7.v1.1,hu1G7.v1.2, hu1G7.v1.3 and hu1G7.v1.4 (FIGS. 2 and 3) were designed.Antibody hu1G7.v1.1 was designed to incorporate the six VL mutationsS30R, I32L, A51G, T56S, H91Q, and S94P. Antibody hu1G7.v1.2 was designedto incorporate the ten VL mutations S30R, I32L, A51G, S52Y, Y53N, T56S,H91Q, Y92F, S93Q, and S94P. Antibodies hu1G7.v1.3 and hu1G7.v1.4 werebased on hu1G7.v1.1 and designed to omit either I32L (hu1G7.v1.3) orH91Q (hu1G7.v1.4).

A focused phage library to investigate mutations of interest (VLmutations S30R, I32L, A51G, S52Y, Y53N, T56S, H91Q, Y92F, S93Q, S94P; VHmutations S28K, L29T, S56T) in many different combinations was alsodesigned. In an effort to further increase similarity to human antibodysequences, the focused phage library also allowed limited variation atframework positions as follows: S43A in the LC, L48I in the HC, and/orN73T in the HC. Approximately 10⁸ transformants were obtained.Selections were performed using FcRH5 protein fragment as “bait”. Theselections were performed initially with solid phase immobilized targetprotein, with solution phase selections used in later rounds. Antibodieshu1G7.v1.5, hu1G7.v1.7, hu1G7.v1.13, and hu1G7.v1.13.1 (FIGS. 2 and 3)were derived from this library.

Sequencing data from experiments with the focused phage library alsosupported the concept (FIGS. 42 and 43) of negative interaction betweenthe I32L and H91Q VL mutations. Following five rounds of selection onphage and subsequent analysis of 163 VL sequences, the frequency ofglutamine at VL position 91 was found to be much higher in the presenceof Ile32 (49/70 clones) than in the presence of Leu32 (5/93 clones).This effect was not observed in the unselected library, in which Gln91was observed in 50% (9/18) of the analyzed clones that contained leucineat position 32. These results suggested negative selection of clonesthat contained both I32L and H91Q, despite positive enrichment of eachof these mutations in other contexts.

Susceptibility to oxidation was investigated by incubating the antibodywith 1 mM 2,2′-azobis(2-methylpropionamidine)dihydrochloride (AAPH) for16 hours, followed by digestion with trypsin to create peptides thatcould be analyzed using liquid chromatography (LC)-mass spectrometry(MS) analysis. For each peptide in the sample, retention time from theLC, and high-resolution, accurate mass and peptide ion fragmentationinformation (amino acid sequence information) were acquired in the MS.Extracted ion chromatograms (XIC) were taken for peptides of interest(native and modified peptide ions) from the data sets at a window of ±10ppm, and the peaks were integrated to determine area. Relativepercentages of modification were calculated for each sample as follows:(area of the modified peptide)/(area of the modified peptide plus thearea of the native peptide)×100. These relative percentages were thencompared between the control and the AAPH-treated samples.

Following a 16-hour incubation at 40° C. in the presence of 1 mM AAPH,an increase in oxidation of HVR-H2 Trp52 and Met64 (44.5% and 52.7%,respectively) was observed. Additionally, the Trp52 oxidation increasedfrom 2% to 62% after light stress, and mutation of Trp-52HC resulted inan affinity loss for 1G7.v1.4 variants, as measured by BIACORE® (FIG.4). Replacements of Trp52 with phenylalanine, tyrosine, leucine, orhistidine and replacements of Met64 with phenylalanine, isoleucine,valine, or leucine were investigated in the context of the light chainsfrom antibodies hu1G7.v1, hu1G7.v1.3, hu1G7.v1.4, hu1G7.v1.5,hu1G7.v1.6, and hu1G7.v1.7 (FIGS. 2 and 3). In particular, mutation ofW52 to F, Y, L, or H resulted in K_(D) values of 119 nM, 131 nM, 92 nM,and 60.4 nM, respectively. While mutation of M64 to V in 1G7.v85resulted in a K_(D) of 2.5 nM. For affinity screening of HVR-H2 Trp52and Met64 variants (Table 5), antibodies were expressed by transienttransfection of Expi293 cells and purified using tip columns that werecustom packed with affinity resin MabSelect SuRe (Glygen Corp.,Columbia, Md. & GE Life Sciences, Piscataway, N.J.). Control antibodieshu1G7.v1 and hu1G7.v1.5 were expressed by transient transfection of 293Tcells and purified with MabSelect SuRe. Binding of FcRH5 proteinfragment to captured antibodies was monitored at 100 nM and 500 nM.Association and dissociation were monitored at a flow rate of 40 μl/min,for 180 s and 300 s, respectively. The results, shown in Table 5,demonstrated that HVR-H2 Met64 can be mutated to valine whilemaintaining high affinity for FcRH5.

Antibody hu1G7.v1.4 was selected based on affinity and selectivity forFcRH5 over other FcRH family members. To improve the resistance of thisantibody to oxidation, the M64V mutation described in Table 5 was alsoincorporated into the VH region. The resulting antibody hu1G7.v1.4.M64Vwas designated hu1G7.v85 (FIGS. 5A-5B) and was produced as a bivalentIgG and as a T cell-dependent bispecific antibody (TDB). The sequence ofhu1G7.v85 is presented in FIGS. 6A-6B and FIG. 5A-5B. Kinetic analysisof hu1G7.v85 is shown in Table 6. Analysis of hu1G7.v1.4 is shown forcomparison. Differences observed in K_(D) of hu1G7.v1.4 (Tables 5 and 6)are within the experimental variation expected for these molecules.

A non-affinity matured version of hu1G7.v.1, hu1G7.v87, was alsoproduced, which possesses the mouse 1G7 HVRs in the humanized frameworkdescribed above, with an M64V mutation to improve oxidation resistance.

TABLE 5 Affinity screening of HVR-H2 position 52 and 54 variants ofhu1G7.v1 Light chain from antibody: Screening K_(D) (M) hu1G7.v1hu1G7.v1.3 hu1G7.v1.4 hu1G7.v1.5 hu1G7.v1.6 hu1G7.v1.7 Heavy None 2.7 ×10⁻⁸ — — 2.0 × 10⁻⁹ — — chain W52F 2.5 × 10⁻⁷ 1.8 × 10⁻⁷ 1.2 × 10⁻⁷ 4.8× 10⁻⁸ 8.1 × 10⁻⁸ 7.7 × 10⁻⁸ mutation W52Y 1.5 × 10⁻⁷ 6.4 × 10⁻⁷ 1.3 ×10⁻⁷ 1.8 × 10⁻⁷ 5.3 × 10⁻⁷ 1.4 × 10⁻⁷ W52L 1.0 × 10⁻⁷ 2.4 × 10⁻⁷ 9.2 ×10⁻⁸ 1.3 × 10⁻⁷ 2.5 × 10⁻⁷ 1.3 × 10⁻⁷ W52H 1.2 × 10⁻⁷ 6.6 × 10⁻⁸ 6.0 ×10⁻⁸ 6.7 × 10⁻⁸ 1.1 × 10⁻⁷ 9.0 × 10⁻⁸ M64I 3.0 × 10⁻⁸ 2.1 × 10⁻⁹ 2.5 ×10⁻⁹ 2.7 × 10⁻⁹ 2.7 × 10⁻⁹ 2.6 × 10⁻⁹ M64V 2.7 × 10⁻⁸ 2.5 × 10⁻⁹ 2.5 ×10⁻⁹ 2.5 × 10⁻⁹ 2.7 × 10⁻⁹ 2.5 × 10⁻⁹ M64L 2.7 × 10⁻⁸ 2.4 × 10⁻⁹ 2.5 ×10⁻⁹ 2.3 × 10⁻⁹ 2.6 × 10⁻⁹ 2.0 × 10⁻⁹ M64F 2.6 × 10⁻⁸ 2.2 × 10⁻⁹ 2.5 ×10⁻⁹ 1.4 × 10⁻⁹ 2.6 × 10⁻⁹ 2.4 × 10⁻⁹

TABLE 6 Kinetic analysis of hu1G7.v1.4 and hu1G7.v85 in hIgG1 and TDBformats Human FcRH5 Cyno FcRH5 Antibody Format K_(D) (nM) Ka (1/Ms) Kd(1/s) K_(D) (nM) Ka (1/Ms) Kd (1/s) 1G7.V1.4 hIgG1 2.6 7 × 10⁵ 2 × 10⁻³7.7 1 × 10⁵ 1 × 10⁻³ TDB 2.4 5 × 10⁵ 1 × 10⁻³ 6.3 1 × 10⁵ 8 × 10⁻⁴1G7.V85 hIgG1 2.5 6 × 10⁵ 2 × 10⁻³ 7.2 1 × 10⁵ 1 × 10⁻³ TDB 2.4 4 × 10⁵1 × 10⁻³ 6.8 1 × 10⁵ 8 × 10⁻⁴

An additional affinity maturation method utilized Sanger sequencing torandomize the heavy chain HVR positions; however, no desirable mutationswere identified by this method. Therefore, the number of positionstested for variants and the depth of screening using next-generationsequencing (Illumina sequencing) were expanded. A library was designedin which selected positions in the heavy chain variable region wererandomized with an NNKcodon, which encodes any amino acid or an amberstop codon. The design allows only one amino acid change in the antibodyvariable regions per clone. The positions were selected from HVRs andframework positions in direct contact or proximal to HVRs. Kabatpositions 1, 2, 24 to 40, 43, 45 to 78, 80 to 83, 85, 86, 91, and 93 to102 were randomized. The library was created by DNA synthesis (GeneWiz),producing 75 independent linear DNA fragments with one position in eachfragment randomized with the NNKcodon. The linear DNA fragments werepooled and cloned into a monovalent Fab fragment phage display vector(Lee et al. J. Immunol. Methods 284:119-132, 2004) containing the lightchain variable region without affinity maturation mutations. Ligationproducts were electroporated into E. coli XL-1, superinfected with M13KO7 helper phage (New England Biolabs) and grown as described. Binderswere selected in three rounds of sorting with human or cynomolgus FcRH5in parallel tracks. For these selections, ELISA plates coated withneutravidin (Pierce) were used to capture biotinylated human orcynomolgus FcRH5 domain 8 at 50 ng/ml in PBS. Phage (1 OD268/ml) wasincubated with immobilized FcRH5 for two hours at room temperature andunbound phage removed by washing 10 times with PBS. Library and selectedphage were used to infect E. coli XL-1, plasmid DNA was extracted andinserts were amplified by an 15-cycle PCR amplification using PhusionDNA polymerase (New England Biolabs) followed by agarose gelpurification of amplicons. DNA from phage from the original library (2OD2₆₈) was extracted with a M13 DNA purification kit (Qiagen) and 200 ngof DNA were used as template to amplify inserts using the sameconditions as above. Amplicons were sequenced by Illumina sequencing aspreviously described (Koenig et al. J. Biol. Chem. 290:21773-21786,2015). Sequences were filtered and analyzed as previously described(Koenig et al. J. Biol. Chem. 290:21773-21786, 2015), removing allsequences with more than one mutation in the variable region, sequenceswith mutations not conforming to the NNKdegenerate codon usage andsequences with stop codons. Enrichment was calculated by dividing thefrequency of a given mutation at a given position in the sorted sampleby the frequency of the very same mutation in the unsorted (initiallibrary) sample, as described previously (Fowler et al. Nat. Methods7:741-746, 2010), using the following formula: Ev=log₂((Rv, s/ΣRx,s)/(Rv, i/ΣRx, i)), where Ev is the enrichment of a mutant, Rv,s is thenumber of reads with mutation v in position s in the sorted population,Rx,s is number of reads in the sorted population with mutations in thesame position as variant Rv,s, Rv,i is the number of reads of the samemutant as Rv,s in the input, unsorted population and Rx,i is the sum ofall variants in the input, unsorted population with mutations in thesame position as variant Rv,i. The Ev scores for selections with humanand cyno FcRH5 are shown in FIGS. 45 and 46, respectively, and the grayboxes represent mutations that were not identified in the selectedsample or in the library.

FIG. 47 shows the mutations with scores of at least 0.5 in selectionswith human FcRH5 and at least 0 in selections with cynomolgus FcRH5. InFIG. 47, the mutations selected for further analysis (i.e., mutationsthat are at least mildly favored for binding to human FcRH5 and that areneutral of better for cynomolgus FcRH5 binding) are highlighted in blackor gray, and the L29T mutation identified by Sanger sequencing ishighlighted in gray. A total of 11 positions had mutations meeting thesecriteria, with multiple variants in some positions. One position had amutation that introduced a glycosylation site (G54N) and was notselected for further characterization. Another variant, L29T, had beenidentified by screening with Sanger sequencing but was only identifiedin clones with the S56T mutation. That variant showed an averageimprovement in affinity of 1.2-fold only. The S56T mutant had a strongnegative enrichment score of −2.1 (FIG. 45), suggesting that the L29Tmutation may have a beneficial effect that was obscured by the presenceof the second S56T mutation in the double mutant. Of the other ninepositions, the mutation with the highest score was selected except forpositions 1 and 65, where two mutations were selected. These variantswere made by DNA synthesis (GenWiz) and expressed as human IgG1 incombination with the light chain of hu1G7.v1 (without affinitymaturation mutations) in Expi293 cells in 1-ml cultures. Human IgG1variants were purified by protein A chromatography and tested inBIACORE® for affinity. Of the two mutations tested, only two, L29T andS100P, had a significant impact on affinity when tested in BIACORE® withsoluble human FcRH5. The L29T mutation alone improved affinity in thatcontext by 2.2-fold, whereas the S100P mutation improved affinity by3-fold. These mutations were tested in isolation and in combination inthe context of an affinity-matured light chain with the I32L, A51G,T56S, and S94P mutations present in hu1G7.v86. The heavy chain L29Timproved the affinity of hu1G7.v86 by about 2-fold, similar to theeffect in the context of a light chain without mutations. In contrast,the S100P, which in the context of a light chain without mutationsimproved the affinity by 3-fold, improved the affinity of hu1G7.v86 by1.5-fold. Combined, the L29T and S100P mutations improved the affinityof hu1G7.v86 by 2.5-fold. Therefore, even using deep mining withnext-generation sequencing and testing many framework positions inaddition to the CDR positions for favored mutations in the heavy chain,only a 2.5-fold improvement in affinity could be achieved. The resultsindicated that mutations that significantly contributed to affinity whentested individually had much less of an impact in molecules whichalready had other mutations. This was particularly true of the S100Pmutation in the heavy chain which, individually, improved affinity ofhu1G7.v1 by about 3-fold but in the context of a molecule with the lightchain mutations I32L, A51G, T56S, and S94P and heavy chain mutation L29Thad little additional impact on affinity (Table 7, compare hu1G7.v91 andhu1G7.v93; hu1G7.v93 sequence presented in FIGS. 7A-7B). Thus, whilesignificant affinity improvements could be achieved by adding severalmutations to the light chain, relatively little improvement could beachieved by introducing mutations into the heavy chain.

TABLE 7 Affinities of hu1G7 variants with mutations identified by deepsequencing of phage display libraries selected with FcRH5 Mutations(Kabat position) Fold improvement over: Variant Heavy Chain Light ChainK_(D) (nM) hu1G7.v1 hu1G7.v86 hu1G7.v1 — — 14.6 — A E1S — 14.6 1.0 B E1F— 19.1 0.8 C S28P — 13.3 1.1 D L29T — 6.7 2.2 E V37Y — 11.0 1.3 F A61P —11.7 1.2 G M64G — 14.4 1.0 H S65D — 15.0 1.0 I S65P — 16.2 0.9 J K81M —15.3 1.0 K S82bD — 15.0 1.0 L S100P — 4.8 3.0 hu1G7.v85 M64V S30R, I32L,A51G, T56S, S94P 0.8 17.0 hu1G7.v86 M64V I32L, A51G, T56S, S94P 1.4 10.3hu1G7.v91 M64V, L29T I32L, A51G, T56S, S94P 0.7 20.9 2.0 hu1G7.v92 M64V,S100P I32L, A51G, T56S, S94P 0.9 15.9 1.5 hu1G7.v93 M64V, L29T, S100PI32L, A51G, T56S, S94P 0.6 25.3 2.5

Example 3. Generation and In Vitro Characterization of Exemplary FcRH5TDBs

Anti-FcRH5 antibodies described herein were used to generate Tcell-dependent bispecific (TDB) antibodies comprising the bindingdeterminants of the anti-human FcRH5 on one arm and an anti-human CD3εon the other arm. The FcRH5 binding determinants included the humanizedand affinity matured monoclonal antibody clones 1G7, 1G7.v85, and1G7.v87. The humanized binding determinants for anti-CD3ε included thehigh-affinity antibody clone 38E4.v1, the high-affinity clone 38E4.v11,and the low-affinity clone 40G5 (EC50 for hCD3ε=1.0 nM, 50 pM, and 13nM, respectively). The anti-CD3 clones 38E4.v1, 38E4.v11, and 40G5c binda human CD3ε polypeptide (a fragment of the human CD3ε polypeptideconsisting of amino acids 1-26 or 1-27 (SEQ ID NO: 174)) and the aminoacid residue Glu5 of CD3ε is not required for binding (see also PCT Pub.No. WO 2015/095392 and U.S. Pub. No. 2015-0166661, each of which isincorporated herein by reference in its entirety).

Exemplary FcRH5 TDBs were characterized to evaluate their therapeuticpotential. Humanized full-length FcRH5 TDB molecules were found to killhuman plasma cells and patient-derived primary myeloma tumor cells atextremely low (pM) doses and to trigger a robust proliferation of Tcells. FcRH5 TDBs were efficacious in suppressing the growth of myelomaxenografts in vivo and resulted in complete depletion of B cells andplasma cells in primates at well tolerated dose levels which wereexpected to saturate the target. Complete plasma cell depletion providedcompelling evidence of efficacy in the bone marrow microenvironment.Activity of the FcRH5 TDBs correlated with target expression (e.g.,FcRH5 expression) level suggesting that high risk myeloma patients withchromosome 1 q copy gain may be uniquely sensitive to thisimmunotherapy.

Materials and Methods

A. Antibodies

a. Production of TDBs

1. Rational Design Approach

To optimize expression and increase the yield of TDBs, mutations wereengineered into the Fc, VH, VL, CH1, and CL domains of the anti-FcRH5antibodies (e.g., FcRH5 TDBs) to drive appropriate antibody monomerformation. In particular, amino acid modifications that introducedcharged regions into the VH, VL, CH1, and/or CL domains provided amechanism to reduce heavy chain and light chain mispairing. Cognatecharged regions having opposite overall charges are driven together andassist in appropriate heavy chain and light chain pairing, increasingthe overall production yield of an anti-FcRH5 antibody (e.g., an FcRH5TDB). Exemplary Rational Design configurations, including amino acidmodifications of the Fc, VH, VL, CH1, and CL domains, are presented inFIGS. 1A-1B.

2. Rosetta Design Approach

In addition, or in the alternative, to optimize expression and increasethe yield of TDBs, mutations were engineered into the Fc, VH, VL, CH1,and CL domains of the anti-FcRH5 antibodies (e.g., FcRH5 TDBs) to driveappropriate antibody monomer formation. The Rosetta Design approachprovided an additional mechanism to drive appropriate heavy chain andlight chain pairing, in addition to charged region mutations, in theform of knob and hole mutations in the CH1 and CL domains. ExemplaryRosetta Design configurations, including amino acid modifications of theFc, VH, VL, CH1, and CL domains, are presented in FIGS. 1C-1F.

3. One-Cell Approach for FcRH5 TDB Production

In one approach, the FcRH5 TDBs can be produced by culturing host cellsthat have been co-transfected with two plasmids, each encoding one ofthe two arms of the FcRH5 TDB (e.g., a first plasmid encoding ananti-FcRH5 half-antibody and a second plasmid encoding an anti-CD3half-antibody). Transfection of host cells (e.g., bacterial, mammalian,or insect cells) was performed in a 96-well plate format. To screen forFcRH5 TDB production, approximately 2,000 to 3,000 clones may be pickedand assessed by ELISA and intact IgG homogeneous time resolvedfluorescence (HTRF) for their ability to bind the target antigen, FcRH5.Clones producing FcRH5 TDBs capable of binding to FcRH5, or a fragmentthereof, were selected for expansion and further screening (e.g., forbinding to CD3). Top clones can then be selected for further analysisbased on percent bispecific antibodies (bsAbs) produced, titer, andperformance qualification (PQ).

An exemplary one-cell approach that can be used to produce FcRH5 TBDs ofthe invention is described in International Patent Application No.PCT/US16/28850, which is incorporated herein by reference in itsentirety.

4. Two-Cell Approach for FcRH5 TDB Production

Alternatively, FcRH5 TDBs can be produced by culturing the antibodyhemimers (e.g., half-antibodies) separately (i.e., in two different celllines) using high-cell density fermentation and then isolating eachhalf-antibody independently by Protein A chromatography. The purifiedhalf-antibodies can then be combined, for example, at a 1:1 molar ratioand incubated in 50 mM Tris, pH 8.5 in the presence of 2 mM DTT for 4hours to allow annealing and the reduction of disulfides in the hingeregion. Dialysis against the same buffer without DTT for 24-48 hoursresulted in the formation of the inter-chain disulfide bonds.

TDBs may be alternatively produced by transfection of two plasmids, eachencoding the distinct arms of the TDB, into separate host cells. Thehost cells may be co-cultured or cultured separately. Transfection ofhost cells may be performed in a 96-well plate format. To screen for TDBproduction, 2,000 to 3,000 clones are picked and assessed by ELISA andintact IgG homogeneous time resolved fluorescence (HTRF) for theirability to bind a selected antigen (e.g., FcRH5). Clones producing TDBscapable of binding to FcRH5, or a fragment thereof, are selected forexpansion and further screening. Top clones are selected for furtheranalysis based on percent bispecific antibodies (bsAbs) produced, titer,and PQ.

5. Exemplary Production Methods

In one example, using this strategy, FcRH5 TDBs were produced by aco-culture strategy using E. coli cells expressing one half-antibody(hole) and E. coli cells expressing the second half-antibody (knob) weregrown together in shaker flasks at a predetermined ratio such that itproduced similar amounts of each half-antibody (see, Spiess et al. Nat.Biotechnol. 31(8):753-8, 2013; PCT Pub. No. WO 2011/069104, which isincorporated herein by reference in its entirety). The co-culturedbacterial broth was then harvested, the cells disrupted in amicrofluidizer and the antibodies purified by Protein A affinity. It hasbeen observed that during microfluidizing and protein A capture the twoarms annealed and formed the hinge inter-chain disulfide bridges.

In another example, full-length bispecific antibodies were produced aspreviously described (Junttila et al. Cancer Res. 74:5561-5571, 2014;Sun et al. Science Trans. Med. 7:287ra270, 2015). Briefly, the twohalf-antibodies (e.g., anti-FcRH5 (e.g., optimized variants of 1G7) andanti-CD3 (e.g., 38E4.v1)) containing “knob” or “hole” mutations in theirCH3 domains were expressed by transient transfection of CHO cells andthen affinity purified with Protein A. Equal amounts of the twohalf-antibodies were incubated with a 200 molar excess of reducedglutathione at pH 8.5 overnight at 32° C. to drive the formation of theknob-hole disulfide bonds. The assembled bispecific antibody (e.g.,FcRH5 TDB) was purified from contaminants through hydrophobicinteraction chromatography. The purified FcRH5 TDBs were characterizedfor purity by mass spectrometry, size exclusion chromatography (SEC),and gel electrophoresis.

b. Purification of FcRH5 TDBs

The FcRH5 TDBs were purified from contaminants by hydrophobicinteraction chromatography (HIC). The resulting material was analyzedfor endotoxin levels using an Endosafe portable test system, and, whenneeded, the endotoxin content was reduced by washing the protein with0.1% Triton X-114. The molecular weights of the TDBs were analyzed bymass spectrometry (LC-ESI/TCF) as described before (Jackman et al. TheJournal of Biological Chemistry. 285:20850-9, 2010). FcRH5 TDBs werealso analyzed by analytical size exclusion chromatography on a ZenixSEC-300 column (Sepax Technologies USA) using an Agilent 1:100 HPLCsystem. The presence of residual antibody fragments were quantified byelectrophoresis using a 2100 Bioanalyzer and a Protein 230 Chip.

c. Labelled Antibodies

All directly labeled antibodies for flow cytometry, except onesotherwise mentioned, were purchased from BD Bioscience. Anti-human PD-1was purchased from Affymetrix. Goat anti-human IgG and goat anti-mouseIgG were purchased from Jackson Immunoresearch. Anti-PC-FITC (cloneVs38c) was purchased from DAKO. SLP-76 antibody for Western Blot waspurchased from Cell Signaling Technology. The p-SLP76 (Ser376) wasgenerated at Genentech, Inc.

For detection of FcRH5 from multiple myeloma (MM) samples and healthydonor plasma and B cells by FACS, anti-FcRH5 antibody 1G7 was labeledwith PE by Southern Biotec. For microscopy, the TDBs were labeled withAlexa Fluor 647 using the appropriate protein labeling kit(ThermoFisher) according to the manufacturer's instructions. TDBs weredialyzed into PBS, pH 7.2 prior to labelling and a dye/protein ratio of4 was routinely achieved.

B. Molecular Stability Assays

To assess molecular stability of the FcRH5 TDBs thermal stress testswere conducted at 30° C. to 40° C. over four weeks. The FcRH5 TDBs wereincubated at 1 mg/mL in 20 mM his-acetate, 240 mM sucrose, pH 5.5, andevaluated after 2 weeks. The TDBs were evaluated for <5% change in Ndeamination/D isomerization, for <2.5% change in monomer loss by SEC,and for <16% in main peak loss by IEC at 2 weeks. The SEC buffer usedwas 0.25M KCl, 0.2M K₃PO₄, pH6.3. LC-MS was conducted in reducedconditions using TCEP at 60° C. for 10 minutes. LC-MS/MS analysis wasconducted by RCM tryptic peptide mapping with DTT reduction, IAAcapping, and pH 8.2 digestion.

An AAPH oxidation assay was conducted to evaluate <35% Trp oxidation and<1.1 Met Ox/Met256. The FcRH5 TDBs were incubated at a concentration of1.0 mg/mL and stressed in 1 mM AAPH for 16 hours. A light oxidationassay was also conducted to evaluate Trp OX/TrpOX_ApoMabW53>0.5. For thelight oxidation assay, the TDBs were incubated at a concentration of 1.0mg/mL for 48 hours and exposed to light for 2.4 million lux hours.Oxidation was evaluated by LC-MS/MS analysis with RCM tryptic peptidemapping with DTT reduction, IAA capping, and pH 8.2 digestion.

C. Cell Culture and Stable Cell Line Generation

HEK-293T and HEK-T cells expressing the 1 G4 TCR complex and key TCRsignaling components (James et al. Nature 487:64-69, 2012) were culturedin DMEM (Sigma Aldrich). All other cell lines were cultured in RPMI. Allmedia was supplemented with 10% heat inactivated FBS (LifeTechnologies), 1 mM HEPES (Lonza), 2 mM glutamine, 100 U/ml penicillinand 100 μg/ml Streptomycin (Sigma Aldrich).

To evaluate the immunological synapse formation, SVT2 cells wereinfected with either retrovirus encoding full-length FcRH5 having anN-terminal gD tag or virus encoding a truncated FcRH5 (i.e., FcRH5including a deletion of amino acids 1-744) having an N-terminal gD tag.To evaluate the target dependency of FcRH5 TDB killing, FOX-NY cellswere infected with lentivirus encoding full-length FcRH5. Single cellderived clones with differential expression levels of FcRH5 were thenselected with 2 μg/ml puromycin.

To evaluate the effect of PD-1/PD-L1 signaling on FcRH5 TDB activity,293 cells were infected with lentivirus encoding FcRH5 followed bytransfection of a plasmid encoding human PD-L1 using Lipofectamine(Invitrogen).

D. Radioligand Cell Binding Assay

The anti-FcRH5 1G7.v85 antibody was iodinated using the lodogen methodto a specific activity of 20 μCi/μg. Competition reaction mixturescontaining a fixed concentration of iodinated antibody and decreasingconcentrations of serially diluted, unlabeled antibody were placed into96-well plates. The cell lines expressing endogenous human FcRH5 (e.g.,MOLP-2, RI-1, KARPAS 620, and KMS21-BM) were washed with binding buffer,which consisted of Dulbecco's Modified Eagle Medium (DMEM) with 2% fetalbovine serum (FBS), 50 mM HEPES (pH 7.2), and 0.1% sodium azide and thenadded to the 96-well plates. The competition reactions with cells wereassayed in triplicate for each concentration of unlabeled antibody andincubated for two hours at room temperature. After the two-hourincubation, the competition reactions were transferred to a MilliporeMultiscreen filter plate (Billerica, Mass.) and washed four times withbinding buffer to separate the free from the bound iodinated antibody.The air-dried filters were counted on a Wallac Wizard 2470 gamma counter(PerkinElmer Life and Analytical Sciences Inc.; Wellesley, Mass.) andthe binding data were evaluated using NewLigand software (Genentech),which uses the fitting algorithm of Munson and Robard to determine thebinding affinity of the antibody (Munson et al. Anal. Biochem.107:22-39, 1980).

E. Vectors and Transient Transfection for Microscopy

FcRH5 having an N-terminal gD tag was fused to the fluorescent proteinmRuby2 by first inserting FcRH5 into the pHR-SIN lentiviral vector,before ligating the mRuby2 DNA sequence into this vector, therebycreating pHR-FcRH5-mRuby2. The SFFV promoter in this vector wassubsequently replaced with the mHSP promoter, creatingpHRI-FcRH5-mRuby2, which utilizes a weaker promoter than pHR, allowingmore physiological expression levels of FcRH5-Ruby. Vectors expressingLCK, ZAP70, CSK/CBP, and CD45 have been described previously (James etal. Nature. 487:64-69, 2012). The CD45 construct used was either the ROisoform or a construct containing the cytoplasmic domain of CD45 withthe transmembrane and extracellular domains of CD43, which is known tomimic the function of CD45. Prior to transfecting constructs, HEK cellswere seeded to approximately 60% confluency in 6-well plates. Vectorswere then transiently transfected at appropriate ratios using GeneJuice(Novagen), following the manufacturer's instructions. Cells were used inexperiments 24-48 hours after transfection.

F. Microscopy Imaging and Analysis

To image cell conjugates, 3×10⁵ cells of each cell type to be imagedwere harvested from culture and resuspended in 100 μl of 20 nM TDB inRPMI-1640 (without Phenol-red). After a 20-30 min incubation to allowcell conjugation, cells were washed with PBS, resuspended in DMEM^(gfp2)imaging medium (Evrogen) and added to 35 mm imaging dishes (Mattek). AnAndor spinning disc confocal microscope system was used to image thecells at 37° C. All images were analyzed and all presented images weremanipulated in an equivalent manner using ImageJ. Presented images werebackground subtracted and then cropped to focus on the pair of cells andthe contrast was optimized. The degree of protein clustering andsegregation was determined by using the intensity of fluorescentlylabelled proteins in the plasma membrane. The plasma membrane wasselected by manually drawing a line and the average fluorescenceintensity of the plasma membrane within the cell-cell interface wasdivided by the average fluorescence intensity of the plasma membraneoutside the cell-cell interface to calculate the degree of clustering orsegregation. To generate an image of the interface of a pair of cellsconjugated by TDBs from a z-stack, the image stack was first deconvolvedand then cropped to highlight the interface region using Huygenssoftware.

G. In Vitro Cytotoxicity and T Cell Activation Assays

Target cells were labeled with carboxyfluorescein succinimidyl ester(CFSE) according to manufacturer's protocol (Life Technology, #C34554).The CFSE-labeled target cells and purified CD8+ cells were mixed in 3:1effector cell to target cell (E:T) ratio and incubated with TDB for 48hours. At the end of the incubation, the cells were analyzed with flowcytometry on a FACSCalibur in automation format. The number of livetarget cells was counted by gating on CFSE+/PI-negative cells. Thepercentage of cytotoxicity was calculated as follows: % cytotoxicity(live target cell number w/o TDB−live target cell number w/TDB)/(livetarget cell number w/o TDB)×100.

a. In Vitro Cytotoxicity Assay Cell Lines

Peripheral blood mononuclear cells (PBMC) and CD8+ separation, CellTiter Glo (Promega), and flow cytometry-based viability assays (48h)were conducted as previously described in Junttila et al. Cancer Res.74:5561-5571, 2014. CD8+ cell were used as effectors in a 3:1effector:target ratio.

b. Human Plasma Cells and Primary Multiple Myeloma Samples

Human bone marrow aspirate of healthy donors (ALLCELLS) were diluted inPBS and bone marrow mononuclear cells (BMMCs) were isolated byconventional gradient separation (Lymphoprep, STEMCELL). A flowcytometry viability assay was used to test the effect of 72h FcRH5 TDBtreatment on BMMC plasma cells. Frozen human BMMCs from MM patients werepurchased from Conversant Bio. Myeloma BMMCs were mixed with freshlyisolated healthy donor CD8+ T cells and the co-culture was treated withFcRH5 TDB for 72h. PI-negative CD38+CD138+ cells were counted by flowcytometry.

H. T Cell Activation and Proliferation Assays

The T cell activation assay has been previously described in Junttila etal. Cancer Res. 74:5561-5571, 2014. Freshly isolated CD8+ T cells werelabeled with CFSE and mixed with target cells (e.g., MOLP-2 cells) in1:1 ratio and co-cultured with 1 μg/ml TDB for 48 hours or five days.Cells were stained with anti-CD8-APC (BD Bioscience, #555634),anti-CD69-PE (BD Bioscience, #555531), and/or anti-CD25-APC (BDBioscience, #555434) and the dilution of fluorescence intensity of CFSEwas analyzed by flow cytometry.

I. Flow Cytometry Analysis for Cyno Plasma Cells

Cyno bone marrow aspirates were diluted (1:10) into ACK lysis buffer(Life Technology, #AI 0492) twice. Cyno bone marrow cells were stainedwith anti-CD45, anti-CD20, and anti-CD38. After wash, cells were fixedand permeabilized with IntraStain kit (DAKO). Cells then were stainedwith anti-PC (Clone Vs38c). The cyno plasma cells were classified byflow cytometry as CD45-CD20-CD38+PC+.

J. ELISA Analysis for Cyno IgG Level

Total cyno serum IgG was quantified using standard colorimetric basedsandwiched ELISA. A goat anti-monkey IgG (Bethyl A140-202A) and ahorseradish peroxidase (HRP) conjugated goat anti-monkey IgG (BethylA140-202P) were used as the capture and detection antibody,respectively. Cyno IgG (Cell Sciences CS120163A) was used as the proteinquantification standard.

K. Western Blot Analysis

Freshly isolated human CD8+ T cells and 293T-FcRH5 cells (2:1 ratio)were treated with 1 μg/ml of 1G7.v85/38E4.v1 (“1G7.v85 TDB”),10A8/38E4.v1 (“10A8 TDB”), or anti-gD/38E4.v1 (“anti-gD TDB”) TDB andwashed in phosphate-buffered saline (PBS) at 4° C. and lysed with RIPAlysis buffer (Cell Signaling Technology). pSLP76 (Ser376) and SLP76 weredetected using standard Western blot methods and antibodies.

Results

A. Binding Affinity of FcRH5 TDBs

Among the FcRH5 TDBs produced were molecules comprising clone 1G7 as theFcRH5-binding domain and selected clones as the CD3-binding domain,including 38E4.v1, 40G5c, and 38.E4.v11. Antibodies hu1G7.v1.1(“1G7.v1.1 TDB”), hu1G7.v1.2 (“1G7.v1.2 TDB”), hu1G7.v1.3 (“1G7.v1.3TDB”), hu1G7.v1.4 (“1G7.v1.4 TDB”), hu1G7.v1.5 (“1G7.v1.5 TDB”),hu1G7.v1.7 (“1G7.v1.7 TDB”), hu1G7.v1.13 (“1G7.v1.13 TDB”), andhu1G7.v1.13.1 (“1G7.v1.13.1 TDB”) were generated as FcRH5 TDBs with ananti-CD3 38E4.v1 arm and determined by BIACORE®, generally as describedherein, to have high affinity for soluble FcRH5 protein fragment (Table8). Expression tests of 1G7.v85 and 1G7.v87 in half-antibody formatsincluding “knob” mutants gave a titer above the disaster bar (FIG. 8).

TABLE 8 Affinity evaluation of eight selected affinity matured variantsin TDB format Variable region category Antibody K_(D) Parental (murinevariable region) 1G7 TDB (mean, n = 3) 5.4 nM Site-directed mutagenesisof 1G7.v1 1G7.v1.1 TDB 2.7 nM 1G7.v1.2 TDB 2.6 nM 1G7.v1.3 TDB 1.5 nM1G7.v1.4 TDB 1.4 nM Focused phage antibody library 1G7.v1.5 TDB 1.1 nM(Library template = 1 G7.v1) 1G7.v1.7 TDB 0.6 nM 1G7.v1.13 TDB 1.2 nM1G7.v1.13.1 TDB 0.9 nM

B. Binding and Cross Reactivity of FcRH5 TDBs

Human FcRH5, cyno FcRH5, human FcRH1, FcRH2, FcRH3, and FcRH4transfected mouse SVT2 cells were used to test binding andcross-reactivity of FcRH5 TDBs. The method was carried out as follows.Cultured SVT2 cells were lifted using non-enzyme cell dissociationbuffer (Sigma, #C5914). 1×10⁵ cells were suspended in 100 μL andincubated with FcRH5 TDBs at 3 μg/ml. Cells were then washed with FACSbuffer (PBS, 1% BSA, 2 mM EDTA) and incubated with goat-anti-human Fc PE(Jackson Immunoresearch, #109-116-170) at a 1:100 dilution. Cells werewashed twice with FACS buffer before flow cytometry analysis was carriedout on a FACSCalibur.

After humanization and affinity maturation of murine 1G7, eight affinitymatured variants in TDB format were generated. The optimized 1G7 TDBswere generated in “knob-into-hole” format with 38E4.v1 as an anti-CD3arm. All eight variants showed a 5- to 15-fold increased affinity overthe murine 1G7 TDB by BIACORE® in the TDB format (Table 8). All variantsdemonstrated negative binding to FcRH1 or FcRH4, with different degreesof positive binding to FcRH2 or FcRH3. The 1G7.v1.4 TDB showed the leastcross-reactivity to FcRH2 and FcRH3 (Table 9). Binding of FcRH5 TDBscontaining different anti-FcRH5 arms (i.e., 1G7, 1G7.v85, or 1G7.v1.4)to FcRH3 was also evaluated by FACS (FIG. 9A).

TABLE 9 FcRH5 TDB Binding and Cross-reactivity SVT2- SVT2- SVT2- SVT2-SVT2- SVT2- Cyno- TDB FcRH1 FcRH2 FcRH3 FcRH4 FcRH5 FcRH5 1G7 − − −− + + 1G7.v1.1 − + + − ++ ++ 1G7.v1.2 − +/− + − ++ ++ 1G7.v1.3 − + + −++ ++ 1G7.v1.4 − +/− +/− − ++ ++ 1G7.v1.5 − + + − ++ ++ 1G7.v1.13 − + +− ++ ++ 1G7.v1.7 − + + − ++ ++ 1G7.v1.13.1 − +/− + − ++ ++

The abilities of 1G7.v1.4, 1G7.v85, and 1G7 TDBs to bind huFcRH5 andhuFcRH3 were evaluated by BIACORE® analysis (FIGS. 10A-10D). Theaffinities of 1G7.v85 TDB and 1G7.v1.4 TDB were comparable. 1G7.v1.4 TDBbound to human FcRH5 and human FcRH3 with a K_(D) of 2.4 nM and 80 nM,respectively, and 1G7.v85 TDB bound to human FcRH5 and human FcRH3 witha K_(D) of 2.4 nM and 90 nM, respectively (FIGS. 10A-10D). Additionalanti-FcRH5 variants hu7D8.L1H2, 17B1-VH66, 17B1, and 15G8 (sequencespresented in FIGS. 11A-13B) were also generated in the TDB format withthe anti-CD3 arm 38E4.v1. The hu7D8.L1H2, 17B1, and 15G8 TDBs weretested for binding to human and cyno FcRH5. Results are presented inFIG. 14 and in Table 4.

Comparison of 1G7.v85 TDB and 1G7.v93 TDB binding to human FcRH5 andcyno FcRH5 was conducted by BIACORE® analysis (FIG. 15). 1G7.v85 TDBexhibited a K_(D) of 2.1 nM for binding to human FcRH5 and a K_(D) of7.8 nm for binding to cyno FcRH5. 1G7.v93 TDB exhibited a K_(D) of 1.1nM for human FcRH5 and 8.1 nm for cyno FcRH5.

Kinetic characterization experiments were carried out (see Table 6). Inthese experiments, binding of soluble FcRH5 protein fragment to purifiedantibodies in human TDB antibody format was analyzed at six differentnon-zero concentrations (a 1:3 dilution series, one concentrationinjected twice as a replicate). Flow rate was set at 40 μl/min andassociation and dissociation monitored for 600 s. Kinetic analysis ofmonovalent 1G7.v85 TDB affinity using a hlgG capture format and 1:1binding model produced a K_(D) for human FcRH5 and cyno FcRH5 of 2.4 nMand 6.8 nM, respectively (FIGS. 16A-16B). Mutation of oxidation proneMet-64HC to Val in the 1G7.v85 TDB did not impact binding affinity.

C. In Vitro Cytotoxicity Assays

To evaluate the function of the humanized FcRH5 TDB variants, cellcytotoxicity on FcRH5 MOLP-2 cells that endogenously express FcRH5 wastested. All variants showed similar target cell killing activity butsignificantly increased activity compared to murine 1G7 (FIGS. 17A-17B).In the case of variant 1G7.v1.4 TDB, the EC50 improved 5- to 13-foldrelative to murine 1G7 TDB (n=10).

Based on its high cytotoxic activity and low cross-reactivity to otherfamily members, 1G7.v1.4 TDB was selected for further analysis. Toincrease the stability of the TDB molecule, the 1G7.v1.4 arm was mutatedat potential oxidization sites, thereby generating a polished version,1G7.v85. The two versions, 1G7.v1.4 and 1G7.v85, were evaluated in theTDB format in various assays, including T cell activation, MOLP-2 targetcell killing activity, cyno B cell in vitro depletion, and cyno plasmacell depletion (FIGS. 18A-18D). In all tested assays, the 1G7.v85 TDBbehaved indistinguishably from the 1G7.v1.4 TDB.

The humanized and polished 1G7.v87 TDB was also compared to the 1G7.v85TDB. Consistent with its lower affinity, the 1G7.v87 TDB exhibitedreduced binding to human FcRH5 relative to the 1G7.v85 TDB as indicatedby flow cytometry analysis (FIG. 19A). 1G7.v87 also showed negativecross-reactivity to FcRH3.

Target cell cytoxicity of the 1G7.v87 TDB and the 1G7.v85 TDB wereevaluated on MOLP-2 CD8+ cells (FIG. 19B) and on PBMCs from four healthydonors (FIG. 19C). In all donors, the 1G7.v87 TDB was negative for humanB cell killing, while thel G7.v85 TDB showed some degree of positivekilling activity. Cyno PBMCs from four healthy donors were also tested(FIG. 19D). The ranking of EC50's of three versions of 1G7 TDBs isconsistent in all three donors. 1G7.v87 TDB is comparable to murine 1G7TDB but has significantly less activity than the 1G7.v85 TDB. The1G7.v87 TDB showed significantly weaker killing activity than the1G7.v85 TDB on cyno B cells in vitro. The 1G7.v85 TDB was also evaluatedfor activity on human NK cells and found to have no killing activity ≤20μg/mL (FIG. 9B). The EC50 of the 1G7.v85 TDB was 5- to 8-fold betterthan that of the 1G7.v87 TDB.

D. Molecular Assessment of Stability of FcRH5 TDBs

Chemical stability of hu1G7.v1 prior to affinity maturation indicatedoxidation susceptibility at Met-64HC and Trp-52_(HC). 1G7.v85 TDBsamples were stressed via AAPH and light stress tests and evaluated forFcRH5 binding by BIACORE®. The 1G7.v85 TDB demonstrated reduced bindingfor FcRH5 after both AAPH and light stress tests as compared to anunstressed control (FIGS. 20A-20D). Monomer stability and chargeheterogeneity of the 1G7.v85 TDB was evaluated by size exclusionchromatography (SEC) and imaged capillary isoelectric focusing (icIEF),respectively, after being stressed in a low pH buffer (his-acetate, pH5.5) for 2 weeks (FIGS. 21A-21B). The observable change in monomer peakloss was small for both monomer stability (0.1%) and chargeheterogeneity (7.7%) (FIGS. 21A-21B). Furthermore, there was noobservable change in the mass of either the light chain or heavy chainsof 1G7.v85 TDB after two weeks of low pH stress (FIGS. 22A-22B). Afterthermal stress at 30° C. for 2 weeks, monomer peak loss was 0.1% by SECand 8% by icIEF.

E. A Membrane Proximal Epitope is Required for Efficient TCR Signalingand Killing Activity of the FcRH5 TDB

To characterize the molecular events that leads to triggering of the Tcell receptor (TCR) upon stimulation by FcRH5 TDB, a reconstitutedsystem was utilized (James et. al. Nature. 487:64-69, 2012) that allowsinitial events leading to receptor activation to be investigated in acontrolled manner. Using healthy donor CD8 cells, the 1G7/UCHT1.v9 TDBresulted in a very robust SLP76 phosphorylation, indicative of TCRsignaling, and mediated efficient killing of target cells (FIGS.23A-23B; EC50=0.5 nM). In contrast, the anti-gD TDB, which targets amembrane-distal epitope, did not result in detectable TCR signaling andwas unable to mediate T cell killing (FIGS. 23A-23B). It was alsoconfirmed that the TDB activity was dictated by the location of theepitope and the size of the extracellular domain by targeting cells thatexpressed heavily truncated target that retained the 1G7 and gD epitopes(FIG. 23C). Activity of the proximal 1G7/UCHT1.v9 TDB increased by25-fold (FIG. 23D; EC50=20 μM), and gD TDB and was able to effectivelymediate killing of cells (EC50=0.19 nM) when the interference caused bythe ECD was removed. Possibility of differential target expression levelbeing the cause for the activity difference between cell lines wasexcluded by FACS analysis (FIG. 24A).

To demonstrate that the differences in the killing activity were relatedto the epitope rather than being properties of the specific antibodyclones, a total of five unique antibody clones targeting the membraneproximal domain for the FcRH5 in TDB format were tested and demonstratedthat the activity of each clone was 20-fold higher compared to 10A8(FIG. 23E). Endogenous expression level of FcRH5 in multiple myeloma islow (e.g., ˜100 to 2000 copies/cell) and comparable to the MOLP-2myeloma cell line (e.g., 2200 copies/cell). When T cells were retargetedto kill MOLP-2 cells only membrane proximal TDBs induced killing of theMOLP-2 cells. Targeting the mid-region of FcRH5 using the 10A8 TDB didnot lead to sufficiently high TCR triggering required for killing ofmyeloma cells (FIG. 24B).

F. FcRH5 TDB Induces Target-Dependent Cell Killing and T CellProliferation

In FIG. 18A, the 1G7.v1.4 TDB and the 1G7.v85 TDB were found to haveindistinguishable T cell-activating abilities. The 1G7.v85 TDB wasevaluated for 1G7 epitope specificity and cyno cross-reactivity (FIGS.25A-25C). The 1G7/38E4.v1 TDB (“1G7 TDB”) bound to MOLP-2 cells, healthydonor B cells, bone marrow plasma cells, and primary myeloma tumor cellsas expected (FIGS. 26A-26D). Preclinical analysis of the 1G7 TDBactivity was initiated by characterization of the mechanism of actionusing healthy donor CD8 cells. 1G7 TDB treatment of target expressingcells resulted in a dose dependent T cell activation (FIG. 27A). Thecytotoxic activity of the 1G7 TDB was exclusive to target positive cellsand correlated with FcRH5 expression level (FIG. 27B). T cell activationby stimulation with 1G7 TDB and target cell led to a robustproliferation of T cells. In five days 95% of the CD8 cells hadundergone as many as six cell divisions as demonstrated by dilution offluorescent dye CSFE (FIGS. 27C-27E). The contribution of the anti-CD3arm and the Fc domain to the cytotoxic activity of the TDB was furtherevaluated, and it was found that a high-affinity anti-CD3 arm wasrequired, while an Fc domain was not required, to achieve cytotoxicactivity (FIGS. 27F-27G).

G. FcRH5 TDB Mediates Potent Killing of Normal Plasma Cells and PatientDerived Primary Myeloma Cells

Expression of FcRH5 in CD138+CD38+ multiple myeloma cells and normalbone marrow plasma cells was studied using the 1G7.v85 TDB and FACS. Allpatient-derived tumor cells and all normal plasma cells expressed FcRH5in all samples suggesting, 100% prevalence in myeloma (FIG. 28A).

Considerable inter-patient variability in expression level was detectedin myeloma. Generally, expression level in tumor cells was notsignificantly elevated compared to normal plasma cells, suggesting thatdeveloping a tumor cell-specific, normal plasma cell-sparing FcRH5 TDBwas likely not feasible. Expression level in normal B cells isconsistently and significantly lower compared to normal plasma cells andmultiple myeloma tumor cells.

The FcRH5 gene is located in the chromosomal breakpoint in 1q21(Hatzivassiliou et al. Immunity. 14:277-289, 2001). Analysis of 20primary multiple myeloma biopsies demonstrated a significant associationbetween FcRH5 RNA expression and 1q21 gain (FIG. 28E), demonstratingthat the chromosomal translocation can lead to FcRH5 overexpression inhigh risk myeloma patients.

The ability of the 1G7.v85 TDB to kill plasma cells was analyzed bytargeting bone marrow mononuclear cells (BMMC) isolated from bone marrowaspirates of healthy donors (FIG. 28B). The 1G7.v85 TDB induceddose-dependent, highly effective, and potent (EC50=85-180 μM) killing ofplasma cells. Similar robust activity was detected when BMMC frommultiple myeloma patients was subjected to 1G7.v85 TDB treatment (FIG.28C). Near 100% killing of myeloma cells was detected with high potency(EC50=60-1200 μM) regardless of the in-life treatment history.

As FcRH5 expression is variable in myeloma (FIG. 28A) and 1G7.v85 TDBactivity correlates with expression level (FIG. 28D), whether patientsin the low end of the expression spectrum would be predicted to respondto FcRH5 TDBs was also investigated. MOLP-2 myeloma cell line wasidentified as a benchmark cell line which has an expression level ofFcRH5 that is similar to average expression in plasma cells and primaryMM cells (FIG. 28A). We also identified several cancer cell lines thatexpress extremely low levels of the target and determined the number ofFcRH5 per cell using Scatchard analysis. The range of FcRH5 bindingsites in these cell lines varied from 2200 to as low as 160 per cell.Despite very low target copy number, the 1G7.v85 TDB induced robustkilling of all tested cell lines (EC50=2-230 μM). Occupancy calculationsindicated that as few as 50 TDB molecules (2% occupancy at MOLP-2;EC50=58 μM) were sufficient to induce T cell activation and target cellapoptosis.

In summary, FcRH5 is expressed in all myeloma patients. FcRH5 TDBs cankill human plasma cells and patient-derived primary myeloma tumor cellsat pM doses. As very few TDBs are required to redirect T cell activity,the molecule potently kills cells with very low expression of thetarget. The results suggest that FcRH5 TDBs have the potential to bebroadly active in myeloma patients and do not support excluding patientsfrom the therapy based on FcRH5 expression level.

H. Cynomolgus Monkey (Cyno) is an Appropriate Safety and Efficacy Modelfor FcRH5 TDB

FACS analysis of peripheral B cells and bone marrow plasma cellsconfirmed that FcRH5 is expressed throughout the B cell lineage in cynosimilar to humans (FIGS. 29A-29B; Poison et al. Int. Immunol.18:1363-1373, 2006). The amino acid sequences of human and cyno FcRH5are 89% identical, and the 1G7.v85 TDB binds to cyno FcRH5 and CD3 withcomparable affinity. In vitro treatment of target cells expressing cynoFcRH5 or MOLP-2 cells expressing human FcRH5 resulted in robust killingusing peripheral T cells from either human or cyno with comparableefficiency (FIGS. 29C-29D). Adding the 1G7.v85 TDB to PBMC/BMMC samplesfrom cyno resulted in a dose dependent and robust killing of cyno Bcells (FIG. 29E) and bone marrow plasma cells (FIG. 29F). In FIG. 18C,the 1G7.v1.4 TDB and 1G7.v85 TDB were found to have indistinguishablecyno plasma cell killing abilities. These results validate cyno as anappropriate safety and efficacy model for anti-FcRH5/CD3.

Example 4. In Vivo Characterization of Exemplary FcRH5 TDBs Materialsand Methods

A. In Vivo Efficacy Studies in Murine Models

a. huNSG/MOLP-2 Mouse Xenograft Model

Female humanized NOD.Cg-Prkdcscid II2rgtm1Wjl/SzJ (NOD/scid gamma; NSG)mice were obtained from The Jackson Laboratory. On the day of cellinoculation, five animals were inoculated with 0.2 mL of MOLP-2 tumorcells at a concentration of 100 million celis/mL, in HBSS/matrigel,subcutaneously in the right flank. As soon as the tumor volumes reacheda volume range of 100-250 mm³, animals were randomized into two groups,a vehicle and treatment group, and the first treatments wereadministered at that time (Day 0). All treatments were administered oncea week by intravenous (i.v.) tail vein injection for a total of fourdoses. The vehicle group was treated with 0.1 ml of 20 mM histidineacetate, pH 5.5, 240 mM sucrose, 0.02% TW-20 buffer. The treatment groupwas treated with 0.1 ml of 1G7.v85 TDB at a concentration of 0.5 mg/kg.Tumors were measured 1-2 times per week with calipers for the durationof the study, and animal body weights were taken at least once a week.For the duration of this study, clinical observations were performedtwice per week to monitor the heaith of the animals.

B. Toxicology Study in Cynomolgus Monkeys

The tolerability, toxicity profile, pharmacokinetics (PK), andpharmacodynamics (PD) of anti-FcRH5 TDB were evaluated in naive, malecynomolgus monkeys (cynos) at Charles River Laboratories (CRL). Cynoswere treated with a single dose, intravenous infusion (1 h) of vehicle,1, 2, or 4 mg/kg 1G7.v85 TDB and were necropsied seven days aftertreatment. The animals were closely monitored for detailed clinicalobservations, respiratory rate, and body temperature during the firstfive hours and at termination. Cage-side clinical observations and bodyweights were collected daily. Blood samples were collected byvenipuncture via the femoral vein pre-study and at selected time pointsthroughout the study for analyses of hematology, serum chemistry,coagulation, and PK total antibody levels) and PD endpoints. PDconsisted of measurements of cytokines (IL-1β, IL-1 RA, IL-2, IL-4,IL-5, IL-6, IL-12/23, IL-13, IL-17, G-CSF, GM-CSF, IFN-γ, TNF-α, andMCP-1), flow cytometry of T-lymphocytes, B-lymphocytes, NK cells,activated T-lymphocytes, PD-1, and circulating cyno IgG. Bone marrow wascollected in anesthetized animals by aspiration from the humeruspre-study and prior to necropsy on Day 8 for evaluation of B-lymphocytesand plasma cells by flow cytometry. At necropsy, organ weights weremeasured and select organs and tissues were thoroughly examined by grossand microscopic examination. Spleen, mesenteric, and mandibular lymphnodes were evaluated for T-lymphocytes, B-lymphocytes, and NK cells. Allprocedures were performed in compliance with the Animal Welfare Act, theGuide for the Care and Use of Laboratory Animals, and the Office ofLaboratory Animal welfare.

C. PKPD Study in Cynomolgus Monkeys and Mice

The concentrations of FcRH5 TDB in serum were determined by genericELISA. Sheep anti-human IgG antibody was used as the capturing reagent,and sheep anti-human IgG conjugated to horseradish peroxidase (HRP) wasused as the detection reagent. Analysis of serum concentration with timefrom available samples were analyzed by a non-compartmental with IVbolus input model (Phoenix™ WinNonlin®, Version 6.3; PharsightCorporation; Mountain View, Calif.). Nominal sample collection time andnominal dose concentrations were used in the data analysis. All TKanalysis was based on individual animal data.

Results

A. FcRH5 TDB Suppresses Growth of Established MOLP-2 Tumors in MiceReconstituted with Human Immune Cells

Modeling anti-myeloma activity of the FcRH5 TDB in mice is challengingsince anti-CD3 antibodies do not cross-react with mouse CD3 and an FcRH5orthologue does not exist in mouse. Therefore, a mouse model with areconstituted human immune system was established by transplanting CD34+selected human hematopoietic stem cells into irradiated mice (huNSGmice). Human CD8+ cells harvested from spleens of huNSG mice were ableto kill MOLP2 cells in vitro with comparable efficiency to humanperipheral CD8+ cells from healthy donors (FIG. 30A). 20 weekspost-transplantation huNSG mice were inoculated with five million MOLP-2cells subcutaneously. Mice with established 100-200 mm³ tumors weretreated with single IV dose of vehicle or 0.5 mg/kg of FcRH5 TDB. FcRH5TDB treatment resulted in tumor regression in all animals (FIG. 30B),indicating that FcRH5 TDB treatment suppresses tumor growth in vivo.

B. FcRH5 TDB has Long Serum Half-Life

A single dose study was designed to evaluate safety, efficacy,pharmacokinetic (PK), and pharmacodynamic (PD) properties of the 1G7.v85TDB in non-human primates. Cynos were treated with a single slowinfusion intravenous dose of vehicle or 1-4 mg/kg 1G7.v85 TDB. Theanimals were closely monitored for adverse effects. Blood samples werecollected at 0, 2, 6, and 24h for cytokine analysis, clinical pathologyanalysis, and PK/PD response. The study was terminated seven days aftertreatment was administered. The FcRH5 TDB demonstrated dose proportionalexposure (Cmax and AUC) between 1-4 mg/kg and was cleared 29-33ml/day/kg in all cohorts (FIG. 31A) and the Cmax at 4 mg/kg dose levelwas 129 μg/ml. This is 2000-fold higher than required to reach in vitrokilling EC50 for human plasma cells and MOLP-2. A PK study was alsoconducted in SCID.bg mice, which are non-binding, and the 1G7.v85 TDBwas found to be have comparable clearance rates as anti-gD TDB (FIG.31B). Receptor occupancy calculations suggested near saturated FcRH5engagement on peripheral blood B cells at Cmax at all dose levels (FIG.32D). These results demonstrated that the 1G7.v85 TDB has a long in vivohalf-life and support a weekly or less frequent dosing schedule.

C. FcRH5 TDB Depletes B Cells and Bone Marrow Plasma Cells in Cyno

FACS analysis of peripheral blood demonstrated a robust pharmacologiceffect on all dose levels. 1G7.v85 TDB treatment resulted in T cellactivation and transient lymphopenia (margination response) within 24hours (FIGS. 31C-31D). B cells remained undetectable in blood seven daysafter the dose, suggesting that they were depleted by the 1G7.v85 TDB(FIG. 31E). In contrast, CD4+ and CD8+ cells recovered by the end of thestudy (FIGS. 32A-32B). All dose levels resulted in complete depletion ofB cells in spleen and bone marrow (FIGS. 31F and 31H). FcRH5 treatmentinduced a robust, dose-dependent depletion of B cells also from lymphnodes (FIGS. 31G and 32C).

Depletion of cyno bone marrow plasma cells in vivo is a key efficacyendpoint in the preclinical development of the anti-FcRH5/CD3. Completedepletion of plasma cells was detected in the animals treated with 2-4mg/kg doses (FIG. 31I). 1G7.v85 TDB treatment also resulted indose-dependent reduction of cyno IgG, an expected secondary outcomeresulting from plasma cell depletion. Theoretically, complete depletionof plasma cells should decrease IgG level ˜30-40% by Day 7. The measuredreduction in cyno IgG was 37% in 2 mg/kg group and 44% in 4 mg/kg group(FIG. 31J). In summary, 1G7.v85 TDBs induced a robust PD response incyno, consistent with its mechanism of action. Complete plasma celldepletion provides compelling evidence of efficacy in the bone marrowmicroenvironment.

D. FcRH5 TDB is Well Tolerated in Cyno

The 1G7.v85 TDB was well tolerated in cyno at ≤4 mg/kg dose levels. Themild/moderate adverse effects that were detected were similar at alldose levels and we failed to see a clear dose response. Clinicalobservations were limited to reversible increases in body temperatureranging from 0.4-1.6° C. within four hours post-dose. Effects onhematology consisted of the expected acute and reversible lymphopeniaattributed to margination. As expected, evidence of an acute andreversible pro-inflammatory state were detected (increased CRP,fibrinogen, prothrombin time, and activated partial thromboplastintime). Treatment caused a reversible increase of ALT, AST, and totalbilirubin.

Consistent with the mechanism of action, the 1G7.v85 TDB induced arapid, generally mild/moderate cytokine release (FIGS. 33A-33F). Alldose levels induced a pro-inflammatory response (including IL-6, IL-5,IFN-g, IL-2, IL-13, G-CSF and MCP-1) and anti-inflammatory response tocounter this (IL1R) peaking at 2-6 h. All cytokines were reversed tonormal level within 24 hours. No signs of extensive or prolongedcytokine release were seen. Extensive histopathological analysisincluding detailed analysis of central nervous system (CNS) did notreveal significant organ toxicity. In summary, maximum tolerated dosewas not reached in the study. The 1G7.v85 TDB was well tolerated at doselevels that are expected to saturate target and sufficient for completedepletion of B cells and plasma cells. No dose response was detected inadverse effects.

Example 5. FcRH5 Combination Therapies

To examine possible FcRH5 combination therapies, a FcRH5 TDB was testedin combination with each of two exemplary PD-1 axis binding antagonists.These experiments demonstrated that, while PD-1/PD-L1 feedback signalingcould reduce FcRH5 TDB-mediated killing, PD-L1 blockade overcame thisinhibition, resulting in improved therapeutic efficacy.

Materials and Methods

A. Antibodies

All labeled antibodies for flow cytometry, except ones otherwisementioned, were purchased from BD Bioscience. The anti-PD-1 antibodyused was KEYTRUDA® (pembrolizumab) and the anti-PD-L1 antibody wasgenerated at Genentech, Inc. Goat anti-human IgG and goat anti-mouse IgGwere purchased from Jackson Immunoresearch. Anti-PC-FITC (clone Vs38c)was purchased from DAKO.

B. PD-1 Induction and Cytotoxicity Assay with Anti-PD-L1

Fresh isolated human CD8+ T cells were mixed with MOLP-2 cells in a 1:1ratio and co-cultured in the presence of 1000 ng/ml of 1G7.v85 TDB for48 hours. The cells were stained with a fluorescein isothiocyanate(FITC)-labelled anti-CD8 antibody (“anti-CD8-FITC”), a phycoerythrin(PE)-conjugated anti-CD69 antibody (“anti-CD69-PE”), and anallophycocyanin (APC)-conjugated anti-PD-1 antibody (“anti-PD-1-APC”),and analyzed by flow cytometry. Cytotoxicity assay of HEK-293T cellsexpressing FcRH5 and PD-L1 (“293-FcRH5-PD-L1 cells”) was set up, asgenerally described herein, with or without 10 mg/ml anti-PD-L1 oranti-PD-1 antibody and analyzed by flow cytometry.

C. Cell Culture and Stable Cell Line Generation

The effect of PD-1/PD-L1 signaling on 1G7.v85 TDB activity was evaluatedby infecting HEK-293T cells with lentivirus encoding FcRH5 followed bytransfection of a human PD-L1 encoding plasmid using lipofectamine(Invitrogen).

D. In Vitro Cytotoxicity and T Cell Activation Assays

Target cells were labeled with carboxyfluorescein succinimidyl ester(CFSE) according to manufacturer's protocol (Life Technology, #C34554).The CFSE-labeled target cells and purified CD8+ cells were mixed in 3:1effector cell to target cell (E:T) ratio and incubated with 1G7.v85 TDBfor 24 to 48 hours. At the end of the incubation, the cells wereanalyzed with flow cytometry on a FACSCalibur in automation format. Thenumber of live target cells was counted by gating on CFSE+/PI-negativecells. The percentage of cytotoxicity was calculated as follows: %cytotoxicity (live target cell number w/o TDB-live target cell numberw/TDB)/(live target cell number w/o TDB)×100.

Results

A. PD-1/PD-L1 Blockade Enhances Activity of the FcRH5 TDB

Strong TCR stimulation signal can lead to immunosuppressive feedbackthat restricts T cell activity. The PD-1/PD-L1 pathway is a criticalcomponent of this feedback and a therapeutically validated immune escapemechanism in several tumor indications. PD-L1 is frequently expressed bymyeloma tumor cells (Gorgun et al. Amer. Assoc. for Cancer Res.21:4607-4618, 2015), and its signaling may limit T cell activity inmyeloma patients. PD-1 is absent in resting T cells, induced upon T cellactivation and limits T cell activity in chronic infection (Zou et al.Science Tran. Med. 8:328rv324, 2016). 1G7.v85 TDB stimulation (48h) ofhuman healthy donor CD8+ cells in the presence of FcRH5-expressing cellsresulted in significant PD-1 induction in T cells (FIG. 34). Thefeedback signal is also activated in vivo. Significant increase inPD-1-positive T cells was seen in cyno T cells at all dose levels. PD-1induction was detected in both CD8+ and CD4+ cells in blood, spleen,lymph nodes, and bone marrow (FIGS. 35A-35B and 36A-36D).

1G7.v85 TDB-mediated target cell killing with primed CD8+ cells wasevaluated in the presence and absence of PD-1/PD-L1 antagonists. Theefficiency of the 1G7.v85 TDB to prime CD8+ T cells to kill PD-L1expressing target cells was modest (FIG. 37A). Blocking PD-1/PD-L1signaling using anti-PD-L1 antibody significantly increased theefficiency of 1G7.v85 TDB-mediated killing (FIG. 37A). In a particularexperiment, primed CD8+ cells were mixed with 293-FcRH5-PD-L1 cells andtreated with the 1G7.v85 TDB alone, or in combination with an either ananti-PD-L1 antibody or the anti-PD-1 antibody (pembrolizumab) (FIG.37B). Combined treatment with an anti-PD-L1 antibody or an anti-PD-1antibody (pembrolizumab) significantly enhanced the efficacy of the1G7.v85 TDB. The EC50 for both combined treatment methods was 0.4 ng/mL,while the EC50 for treatment with 1G7.v85 TDB alone was 0.63 ng/mL (FIG.37B).

These results demonstrate that 1G7.v85 TDB-mediated activation of Tcells leads to induction of PD-1 in T cells in vitro and in vivo. Theseresults further demonstrate that PD-1/PD-L1 signaling can limit FcRH5TDB-mediated killing and that PD-L1 blockage can overcome thisinhibition and lead to improved efficacy. These data support the use ofFcRH5 TDB in combination with a PD-1 axis binding antagonist, such as ananti-PD-1 antibody or an anti-PD-L1 antibody.

B. Dexamethasone Reduces the First-Dose Cytokine Response withoutAffecting FcRH5 TDB Activity

Target cell killing was also evaluated in the presence and absence ofdexamethasone (Dex), a component of standard of care in myeloma that hasanti-inflammatory and immunosuppressant effects (FIG. 38A). The IC50 forthe 1G7.v85 TDB in buffer or in DMSO was 7 μM and 6 μM, respectively. Inthe presence of 0.1 μM or 1 μM Dex, the IC50 for the 1G7.v85 TDB was 16μM and 25 μM, respectively. Dex combination treatment had only a modesteffect on 1G7.v85 TDB efficacy and significantly reduced IL-2, IL-6,TNF-α, and IFN-γ levels. These results demonstrate that dexamethasonemay be used in combination with FcRH5 TDB therapy to mitigate afirst-dose cytokine response in patients (FIG. 38B).

Example 6. Production and Testing of FcRH5 Bis-Fabs

A. Preparation of Thio-Fabs and Hinge-Cys-Fabs and Protein Production

To prepare antibody fragments with free sulfhydryl groups, cysteine(Cys) substitutions were introduced into antibody constructs at variouspositions in either the variable or the constant domains of light chainsor heavy chains by site-directed mutagenesis to create thio-mAbs, asdescribed previously in Junutula et al. J. Immunol Methods332(1-2):41-52, 2008. Thio-Fabs were generated enzymatically fromthio-mAbs by diluting thio-mAbs to 1 mg/mL in 25 mM Tris, pH 8.0,followed by enzymatic digestion at 37° C. for 1 hour using Lys-C(WakoChemicals USA, Inc., Richmond, Va.) at a 1:1000 (wt:wt) ratio of enzymeto antibody. The Lys-C digestion was stopped with 51aM of the proteaseinhibitor tosyl-L-lysine chloromethyl ketone (TLCK) (Bachem, Torrence,Calif.) and purified by cation ion exchange chromatography on a 5 mLHi-Trap SP FF column (GE Healthcare, Piscataway, N.J.) using a 50-mMsodium acetate buffer and a 0-300-mM NaCl 10 column volume (CV)gradient. The thio-Fabs produced by this method are sometimes referredto as “enzymatic thio-Fabs” herein. In another approach, DNA constructsencoding Fabs having an engineered Cys residue or DNA constructsencoding heavy chain fragments containing one native Cys residue in thehinge region, were subcloned into plasmid expression vectors andexpressed directly in CHO cells. The thio-Fabs produced by this methodare sometimes referred to as “recombinant thio-Fabs” herein. A thirdapproach was used for antibodies lacking an engineered Cys residue andrelied upon the native Cys residue(s) present in the hinge region ofIgG. This method is used to produce “hinge-cys-Fabs” and is described infurther detail below.

For the preparation of hinge-cys-Fabs from native antibodies that do notcontain an engineered cysteine for use in synthesis reactions, thefollowing enzymatic procedure, as depicted in panel 1 of FIG. 39, wasused. Both FcRH5 and CD3 parental antibodies were digested with pepsin(1% w/w) treatment in sodium acetate buffer at pH 4.5. After digestionfor 1 hour, the F(ab′)₂ was isolated from the digestion mixture bycapture on an SP-HP cation exchange resin and purified by a 10 CV saltgradient of 0-1 M NaCl. The F(ab′)₂ was then reduced in a buffercontaining 25 mm MES, pH 5.8, 2 mM EDTA, and 300 mM NaCl. Afterreduction with 1 mM TCEP, the Fabs were oxidized, as depicted in panel 2of FIG. 39, by the addition of 5 mM DHAA to reform the disulfide betweenthe heavy chain and light chain. It was routinely observed that, underthese reaction conditions, only the disulfide between the heavy chainand light chain was reformed; the two cysteine residues in the hingeregion remained unoxidized.

The two free thiols (Cys residues) at the hinge, as depicted in panel 2of FIG. 39, were then reacted with a 1M equivalent of N-ethylmaleimide(NEM) (Sigma Aldrich, St. Louis, Mo.). The resultant mixture containingsingly-modified, doubly-modified, and unmodified Fabs was then reacted,as depicted in panel 3 of FIG. 39, with an excess of the bis-maleimidecrosslinker.

These reaction conditions yielded three products: Fabs with onecrosslinker and one NEM, Fabs with two NEM, and Fabs containing only onecrosslinker. The Fabs containing only one crosslinker were found to haveno free cysteine. Thus, under these reaction conditions, a singlecrosslinker reacted very efficiently with both cysteines resulting in amolecule in which the cysteines had been cyclized by the crosslinker.The material comprising the above three reaction products was purifiedfrom the reaction mixture (to remove unwanted reaction components) bygel filtration and used in coupling to other hinge-cys-Fabs, as depictedin panel 4 of FIG. 39, prepared in a similar manner or to thio-Fabs.Only hinge cys-Fabs or thio-Fabs prepared as described and containingone crosslinker, one free maleimide, and one free sulfhydryl were ableto react in the bis-Fab synthesis reactions described in detail below.

B. Protein Expression and Purification

To facilitate purification, Fabs were expressed with either a Flag- orHis-tag. Expression in CHO cells was carried out by standard procedures.Affinity purification following cell culturing was carried out usinganti-Flag mAb resin or Nickel beads resin. Purified thio-Fabs werecharacterized by SDS-PAGE and mass spectrometry. These characterizationsoften showed mass increases of 275 Da and 306 Da. These mass increaseswere found to be disulfide adducts on the unpaired cysteine which wereremoved by reduction and oxidation to prepare the thio-Fabs forcrosslinking with bis-maleimide. The reduction and oxidation ofthio-Fabs was carried out as follows. First, thio-Fabs were reduced for24 hrs by the addition of 2 mM tris(2-carboxyethyl) phosphine HCl(TCEP-HCl; also referred to as TCEP) (Pierce [Thermo Fisher Scientific],Rockford, Ill.) in a buffer containing 25 mM MES, pH 5.8, 300 mL NaCl,and 5 mM EDTA. After reduction, the protein was oxidized by the additionof 5 mM dehydroascorbic acid (DHAA) (Sigma-Aldrich, St. Louis, Mo.). Theisolated thio-Fabs were analyzed by SDS-PAGE and mass spectrometry toensure that the proteins are properly reduced and oxidized.

C. Bis-Fab Synthesis

Two different types of crosslinkers could be used to covalently link thetwo Fabs: bis-maleimide and the pair of adaptersDBCO-PEG-malemide/bromoacetamide-PEG-Azide.

Conjugation Using Bis-Maleimide Crosslinkers

In the first stage of the bis-Fab synthesis, thio-Fabs or hinge-cys-Fabswith an unpaired cysteine were used. Generally, the thio-Fab orhinge-cys-Fab was in the same buffer in which the reduction (FIG. 39,panel 1) and oxidation (FIG. 39, panel 2) was carried out (MES, pH 5.8,2 mM EDTA, and 300 mM NaCl) at a protein concentration of 1 mg/mL. Therewere two potential undesired reaction products at this stage: disulfidedimers and crosslinked dimers. Having a protein concentration of 1 mg/mLat this stage of the synthesis was an important feature of the reactionbecause dimerization was minimized at that protein concentration. Inaddition, controlling the reaction by using a low pH buffer with EDTAhelped minimize dimerization.

A five-fold excess of bis-maleimide crosslinker (Quanta BioDesign,Powell, Ohio) was added to the reaction mixture, as depicted in panel 3of FIG. 39. This five-fold excess of crosslinker was also helpful inminimizing undesirable dimerization. The reaction was incubated at roomtemperature (RT) or 37° C. for four hours until complete. The mixturewas then concentrated to a volume suitable for gel filtration. A 22 mLS-200 Tricom column (GE Healthcare, Piscataway, N.J.) for ag to mgquantity synthesis was used. This first gel filtration step allowed forthe removal of unused crosslinker yielding a purified thio-Fab orhinge-cys-Fab conjugated to the crosslinker. The conditions describedabove typically resulted in at least 90% or greater of the desiredproduct. No thio-Fab or hinge-cys-Fab remained as free-thiol as all wereconjugated to either a crosslinker or bound by disulfide to anotherthio-Fab or hinge-cys-Fab through the unpaired cysteines. The isolatedand purified thio-Fab (or hinge-cys-Fab) plus crosslinker species wasthen added to the second thio-Fab (or hinge-cys-Fab) and concentrated to5 mg/mL or greater, generally to a volume suitable for gel filtration,as depicted in panel 4 of FIG. 39. A protein concentration of at least 5mg/mL during this stage of the synthesis was important to drive thereaction to completion. Lower protein concentrations resulted information of only small quantities of crosslinked bis-Fab dimers.Without being bound by theory, it was hypothesized that a steric effector viscosity-related variable that hindered formation of cross-linkedbis-Fab dimers was overcome by increasing concentrations of reactants.In addition, a range of protein concentrations up to and including 65mg/mL was tested. A correlation between protein concentration andreaction time was found such that the higher the protein concentration,the faster the reaction reached completion. After 2-24 hours at roomtemperature or 37° C., the reaction was complete as determined by massspectrometry. Generally, one reagent was in excess and remaineduncoupled in the final mixture.

Conjugation Using DBCO-PEG-Malemide/Bromoacetamide-PEG-Azide

One of the purified and deblocked Fabs was reacted in 5 molar excess ofDBCO-PEG-malemide (#760676, Sigma) in 50 mM HEPES pH 8 while the otherFab was reacted with 5 molar excess of Azide-PEG-maleimide (#21097BroadPharm) in 50 mM HEPES pH 8. After a one-hour incubation at 37° C.,the reaction was checked by mass spectrometry to verify completion ofthe reaction. The conjugated Fabs were purified from the excess ofcrosslinker by SEC and subsequently mixed at a 1:1 ratio and adjusted toa concentration above 5 mg/ml and incubated overnight at roomtemperature.

Regardless of the crosslinker used, the completed reaction was againpurified by gel filtration; this time the dimeric peak was collected,which contained the 100 kD bis-Fab irreversibly crosslinked through thefree cysteine amino acid (in the case of thio-Fabs) or through theunpaired cysteine located in the hinge region (in the case ofunengineered hinge-cys-Fabs). The reaction progress during both stepswas often monitored by mass spectrometry which clearly showed thepresence of both reactants and the formation of the bis-Fab product. Thepurity of the desired product after the second gel filtration wasdetermined by mass spectrometry and SDS-PAGE. Upon reduction andSDS-PAGE analysis, irreversible crosslinking was observed by thepresence of a 50-kD band representing non-reducible crosslinked chains.Using the process described above at small scale, microgram yields withmicrogram quantities of starting materials were typically achieved. Inaddition, at a larger scale, milligram yields from milligram quantitiesof starting materials were typically achieved.

D. Synthesis of Bis-Fabs Targeting CD3 and FcRH5

Bispecific bis-Fabs obtained from two different antibodies that targetCD3 and FcRH5 were generated. The anti-CD3 parent antibody used could beany anti-CD3 antibody, such as 38E4v.1, 38E4.v11, or 40G5. In oneembodiment, the bis-Fab utilizes 38E4.v1 as the anti-CD3 component, witha light chain sequence of SEQ ID NO: 134 and a heavy chain sequence ofSEQ ID NO: 133. The anti-FcRH5 antibody parent antibody used can also beany anti-FcRH5, for example, those antibodies described herein such ashu1G7.v85 and hu1G7.v87. Specifically, one exemplified bis-Fab includesa variable light chain sequence of SEQ ID NO: 105 and a variable heavychain sequence of SEQ ID NO:104.

For each of these antibodies, recombinant thio-Fabs were produced in CHOcells as described above. Then bis-Fabs were synthesized from thethio-Fabs in a combinatorial format using a synthesis matrix, startingwith approximately 2 mg of each thio-Fab. The different thio-Fabs werecombined to synthesize four unique bis-Fab molecules. Approximately onemg of each bis-Fab was recovered from the synthesis for the shownexamples but yields were expected to vary depending on the differentthio-Fabs. Each of the bis-Fabs was given a unique identifier. Thepurity of each bis-Fab was analyzed by SDS-PAGE and mass spectrometryusing standard methods well known in the art.

Using the matrix recombination approach described above, a series ofCD3- and FcRH5-derived bis-Fab structural variants was synthesized. Fourdifferent thio-attachment points were chosen to synthesize the bis-Fabs;one of the positions was in the heavy chain of the anti-CD3 thio-Fab arm(e.g., at position 76 (Cys76_(HC))), one position was in the light chainof the anti-CD3 thio-Fab arm (e.g., at position 22 (Cys22_(LC))), one ofthe positions was in the heavy chain of the anti-FcRH5 thio-Fab arm(e.g., at position 114 (Cys114_(HC))), and one position was in the lightchain of the anti-FcRH5 thio-Fab arm (e.g., at position 149(Cys149_(LC))). Other positions can be utilized for the insertion of therequired cysteines. Fabs containing thio-attachment points were derivedfrom three different sources; (1) thio-mAbs with cysteine substitutionsthat were digested with Lysine-C to liberate the thio-Fab from theantibody, (2) thio-Fabs with cysteine substitutions that were directlyexpressed in and purified from CHO cells, and (3) hinge-cys-Fabsgenerated by the enzymatic method described above for the attachment ofa single crosslinker to the hinge region of a non-engineered antibodyafter digestion with pepsin. This approach resulted in differentsubstitution points in thio-Fabs for recombination with other thio-Fabs,thus yielding structural variants (see Table 10).

TABLE 10 Position of the engineered Cys in each Fab and correspondingbis-Fab numbers FcRH5 Fab (1G7.v85) HC A114C LC A149C Hinge Cys CD3 FabHC S76C bis-Fab A bis-Fab C (38E4.v1) LC N22C bis-Fab B bis-Fab D HingeCys F(ab′)₂ A

E. Biological Activity of FcRH5 Bis-Fabs

Next, each of the bis-Fab structural variants was tested for theirbinding ability to each of the antigens (i.e., CD3 and FcRH5) by ELISA.The bis-Fab generated by linking the Cys in the hinge region resembledthe architecture of a natural antibody and served as a control. Whileall bis-Fab constructs had similar binding capability to FcRH5 (FIG.40A), most of the bis-Fabs showed reduced binding to CD3 compared to thereference bis-Fab (FIG. 40B). The biological activity was evaluated inan in vitro T cell activation assay that served as a surrogate for Tcell killing activity (FIG. 41). This assay used a Jurkat cell line(Jurkat-Dual, Invivogen) stably transfected with a luciferase reporterunder the control of the transcription factor NF-kB, and the advantageof using this assay over a T cell killing assay was that the number ofcells could be utilized was unlimited. In PBMC cell killing assays, thenumber of tests was limited by the number of cells that could beobtained from a single donor. The assay was carried out as follows. Anappropriate cell line, such as MOLP-2, was chosen as the target cell andco-cultured with Jurkat cells. 10,000 cell line target cells and 50,000effector cells (Jurkat) were added per well (10,000 target cells perwell, 200 μL total volume, with ratio of target:effector=1:5), with orwithout the presence of bis-Fabs. After overnight incubation, 10 μl ofthe supernatant of the different wells was assayed for Luciferaseactivity using 50 μL of QUANTI-LUC (Invivogen), and luminescence wasquantified in an Envision (Perkin Elmer) luminometer instrument.

Interestingly, although the HVR sequences of each bis-Fab variantgenerated for the anti-CD3 or the anti-FcRH5 were not altered in thegeneration of the bis-Fabs themselves, the maximum amount of T-cellstimulation observed for each bis-Fab showed variance, one bis-Fab fromanother, simply based upon the position of the cysteine engineeredcross-linkage. Without being bound by theory, this may have implicationsfor how toxicity and/or potency of a bis-Fab may be modulated to suit aparticular therapeutic or diagnostic need. Another observation was thatsome bis-Fabs (e.g., bis-Fab C) had a significantly reduced affinitytowards CD3, yet had a T cell activating activity comparable to that ofthe reference F(ab′)₂ A (FIGS. 40B and 41, Table 10).

F. Biological Activity of FcRH5 Bis-Fabs Using Endogenous Human B Cells

Each bis-Fab variant was tested for its biological efficacy in a T celldependent bis-Fab killing assay for peripheral endogenous B cellkilling, as follows: 200,000 hPBMCs isolated from each of three healthydonors were added per well with or without bis-Fabs. After a 48-hrincubation, cells were stained with an appropriate cell surface antigenof the target cell line (B cells=CD20) (5 μl/well) and propidium iodidefor cell viability assessment and then analyzed by FACS. Thebis-Fab-dependent killing activities were calculated according to thefollowing equation: % killing=(1−number of live cells withbis-Fabs/number of live cells without bis-Fabs)×100. As a positivecontrol, an F(ab′)₂ was used which was derived from a “knob into hole”FcRH5 TDB antibody wherein the anti-CD3 and anti-FcRH5 arms of each Fabhad the same sequence as those used for the bis-Fabs tested (exceptwithout the cysteine-engineered point mutations) (see e.g., Ridgway etal. Protein Eng., 9:617-621, 1996). Generally, results demonstratedreproducibility, despite using different donor cells.

The amounts of FcRH5 bis-Fabs tested herein required for half-maximallysis of the peripheral endogenous human B cells, or EC50 potency valueexpressed in ng/ml, was calculated for each bis-Fab tested above.Overall, it was expected that trends in potency for each bis-Fab testedin the endogenous human B cell assay would track results determined inthe ELISA assay described above (FIGS. 40A-40B).

1-335. (canceled)
 336. One or more isolated nucleic acids encoding ananti-FcRH5 antibody, wherein the anti-FcRH5 antibody comprises a bindingdomain that binds to FcRH5, the binding domain comprising the followingsix HVRs: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:1; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 8; (c)an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 9; (d) anHVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (e) anHVR-L2 comprising the amino acid sequence of SEQ ID NO: 16; and (f) anHVR-L3 comprising the amino acid sequence of SEQ ID NO:
 23. 337. The oneor more isolated nucleic acids of claim 336, wherein the binding domaincomprises (a) a heavy chain variable (VH) domain comprising an aminoacid sequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO: 104; (b) a light chain variable (VL) domaincomprising an amino acid sequence having at least 95% sequence identityto the amino acid sequence of SEQ ID NO: 105; or (c) a VH domain as in(a) and a VL domain as in (b).
 338. The one or more isolated nucleicacids of claim 336, wherein the anti-FcRH5 antibody further comprisesthe following heavy chain variable region framework regions (FRs): (a)an FR-H1 comprising the amino acid sequence of SEQ ID NO: 52; (b) anFR-H2 comprising the amino acid sequence of SEQ ID NO: 54; (c) an FR-H3comprising the amino acid sequence of SEQ ID NO: 46; and (d) an FR-H4comprising the amino acid sequence of SEQ ID NO:
 47. 339. The one ormore isolated nucleic acids of claim 338, wherein the VH domaincomprises the amino acid sequence of SEQ ID NO:
 104. 340. The one ormore isolated nucleic acids of claim 336, wherein the anti-FcRH5antibody further comprises the following light chain variable regionFRs: (a) an FR-L1 comprising the amino acid sequence of SEQ ID NO: 48;(b) an FR-L2 comprising the amino acid sequence of SEQ ID NO: 57; (c) anFR-L3 comprising the amino acid sequence of SEQ ID NO: 50; and (d) anFR-L4 comprising the amino acid sequence of SEQ ID NO:
 51. 341. The oneor more isolated nucleic acids of claim 340, wherein the VL domaincomprises the amino acid sequence of SEQ ID NO:
 105. 342. One or moreisolated nucleic acids encoding an anti-FcRH5 antibody, wherein theanti-FcRH5 antibody comprises a binding domain that binds to FcRH5, thebinding domain comprising a binding domain comprising (a) a VH domaincomprising the amino acid sequence of SEQ ID NO: 104 and (b) a VL domaincomprising the amino acid sequence of SEQ ID NO:
 105. 343. The one ormore isolated nucleic acids of claim 336, wherein the anti-FcRH5antibody comprises a substitution mutation reducing the effectorfunction of the anti-FcRH5 antibody or FcRH5-binding antibody fragmentthereof.
 344. The one or more isolated nucleic acids of claim 343,wherein the substitution mutation is at amino acid residue N297, L234,L235, D265, and/or P329 (EU numbering).
 345. The one or more isolatednucleic acids of claim 344, wherein the substitution mutation isselected from the group consisting of N297G, N297A, L234A, L235A, D265A,and P329G.
 346. The one or more isolated nucleic acids of claim 345,wherein the substitution mutation is an N297G substitution mutation.347. The one or more isolated nucleic acids of claim 336, wherein theanti-FcRH5 antibody is a monoclonal, human, humanized, or chimericantibody.
 348. The one or more isolated nucleic acids of claim 336,wherein the anti-FcRH5 antibody is an IgG antibody.
 349. The one or moreisolated nucleic acids of claim 336, wherein the anti-FcRH5 antibody isan antibody fragment that binds FcRH5.
 350. The one or more isolatednucleic acids of claim 349, wherein the antibody fragment is selectedfrom the group consisting of bis-Fab, Fab, Fab′-SH, Fv, scFv, and(Fab′)₂ fragments.
 351. The one or more isolated nucleic acids of claim336, wherein the anti-FcRH5 antibody is a full-length antibody.
 352. Theone or more isolated nucleic acids of claim 336, wherein the anti-FcRH5antibody is a multispecific antibody.
 353. The one or more isolatednucleic acids of claim 352, wherein the multispecific antibody is abispecific antibody.
 354. The one or more isolated nucleic acids ofclaim 353, wherein the bispecific antibody comprises a second bindingdomain that binds cluster of differentiation 3 (CD3).
 355. The one ormore isolated nucleic acids of claim 354, wherein the second bindingdomain binds to an epitope on CD3 comprising amino acid residue Glu6 ofCD3.
 356. The one or more isolated nucleic acids of claim 355, whereinthe second binding domain is capable of binding to a human CD3polypeptide or a cyno CD3 polypeptide.
 357. The one or more isolatednucleic acids of claim 354, wherein the second binding domain comprisesthe following six HVRs: (a) an HVR-H1 comprising the amino acid sequenceof SEQ ID NO: 115; (b) an HVR-H2 comprising the amino acid sequence ofSEQ ID NO: 116; (c) an HVR-H3 comprising the amino acid sequence of SEQID NO: 117; (d) an HVR-L1 comprising the amino acid sequence of SEQ IDNO: 118; (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:119; and (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:120.
 358. The one or more isolated nucleic acids of claim 354, whereinthe second binding domain comprises the following six HVRs: (a) anHVR-H1 comprising the amino acid sequence of SEQ ID NO: 115; (b) anHVR-H2 comprising the amino acid sequence of SEQ ID NO: 116; (c) anHVR-H3 comprising the amino acid sequence of SEQ ID NO: 121; (d) anHVR-L1 comprising the amino acid sequence of SEQ ID NO: 118; (e) anHVR-L2 comprising the amino acid sequence of SEQ ID NO: 119; and (f) anHVR-L3 comprising the amino acid sequence of SEQ ID NO:
 123. 359. One ormore isolated nucleic acids encoding an anti-FcRH5 antibody that bindsto FcRH5 and CD3, wherein the anti-FcRH5 antibody comprises ananti-FcRH5 arm comprising a first binding domain comprising thefollowing six HVRs: (a) an HVR-H1 comprising the amino acid sequence ofSEQ ID NO: 1; (b) an HVR-H2 comprising the amino acid sequence of SEQ IDNO: 8; (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 9;(d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (e)an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 16; and (f)an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23; and ananti-CD3 arm comprising a second binding domain comprising the followingsix HVRs: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:115; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 116;(c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121; (d)an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 118; (e) anHVR-L2 comprising the amino acid sequence of SEQ ID NO: 119; and (f) anHVR-L3 comprising the amino acid sequence of SEQ ID NO:
 123. 360. Theone or more isolated nucleic acids of claim 354, wherein the anti-FcRH5antibody or FcRH5-binding antibody fragment thereof comprises one ormore heavy chain constant domains, and wherein the one or more heavychain constant domains are selected from a first CH2 domain (CH2₁), afirst CH3 domain (CH3₁), a second CH2 domain (CH2₂), and a second CH3domain (CH3₂).
 361. The one or more isolated nucleic acids of claim 360,wherein at least one of the one or more heavy chain constant domains ispaired with another heavy chain constant domain.
 362. The one or moreisolated nucleic acids of claim 361, wherein the CH3₁ and the CH3₂ eachcomprise a protuberance (P₁) or a cavity (C₁), and wherein the P₁ or theC₁ in the CH3₁ is positionable in the C₁ or the P₁, respectively, in theCH3₂.
 363. The one or more isolated nucleic acids of claim 362, whereinthe CH3₁ and the CH3₂ meet at an interface between the P₁ and the C₁.364. The one or more isolated nucleic acids of claim 362 or 363,wherein: (a) the P₁ comprises a T366W substitution mutation (EUnumbering); and/or (b) the C₁ comprises T366S, L368A, and Y407Vsubstitution mutations (EU numbering).
 365. One or more isolated nucleicacids encoding an anti-FcRH5 antibody that binds to FcRH5 and CD3,wherein the anti-FcRH5 antibody comprises an anti-FcRH5 arm comprising afirst binding domain comprising the following six HVRs: (a) an HVR-H1comprising the amino acid sequence of SEQ ID NO: 1; (b) an HVR-H2comprising the amino acid sequence of SEQ ID NO: 8; (c) an HVR-H3comprising the amino acid sequence of SEQ ID NO: 9; (d) an HVR-L1comprising the amino acid sequence of SEQ ID NO: 12; (e) an HVR-L2comprising the amino acid sequence of SEQ ID NO: 16; and (f) an HVR-L3comprising the amino acid sequence of SEQ ID NO: 23; and an anti-CD3 armcomprising a second binding domain comprising the following six HVRs:(a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 115; (b)an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 116; (c) anHVR-H3 comprising the amino acid sequence of SEQ ID NO: 121; (d) anHVR-L1 comprising the amino acid sequence of SEQ ID NO: 118; (e) anHVR-L2 comprising the amino acid sequence of SEQ ID NO: 119; and (f) anHVR-L3 comprising the amino acid sequence of SEQ ID NO: 123; and whereinthe anti-FcRH5 arm and the anti-CD3 arm each comprise an N297Gsubstitution mutation (EU numbering); and wherein the anti-FcRH5 armcomprises a T366W substitution mutation (EU numbering) and the anti-CD3arm comprises T366S, L368A, and Y407V substitution mutations (EUnumbering).
 366. One or more vectors comprising the one or more isolatednucleic acids of any one of claims 336, 359, and
 365. 367. One or morehost cells comprising the one or more vectors of claim
 366. 368. The oneor more host cells of claim 367, wherein the one or more host cells areone or more prokaryotic host cells.
 369. The one or more host cells ofclaim 368, wherein the one or more prokaryotic host cells are one ormore E. coli host cells.
 370. The one or more host cells of claim 367,wherein the one or more host cells are one or more mammalian host cells.371. The one or more host cells of claim 370, wherein the one or moremammalian host cells are one or more Chinese hamster ovary (CHO) hostcells.
 372. A method of producing an anti-FcRH5 antibody, the methodcomprising culturing the one or more host cells of claim 367 in aculture medium.
 373. The method of claim 372, wherein the method furthercomprises recovering the anti-FcRH5 antibody from the one or more hostcells or the culture medium.